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Sample records for regulator rcac require

  1. Informational requirements for transcriptional regulation.

    PubMed

    O'Neill, Patrick K; Forder, Robert; Erill, Ivan

    2014-05-01

    Transcription factors (TFs) regulate transcription by binding to specific sites in promoter regions. Information theory provides a useful mathematical framework to analyze the binding motifs associated with TFs but imposes several assumptions that limit their applicability to specific regulatory scenarios. Explicit simulations of the co-evolution of TFs and their binding motifs allow the study of the evolution of regulatory networks with a high degree of realism. In this work we analyze the impact of differential regulatory demands on the information content of TF-binding motifs by means of evolutionary simulations. We generalize a predictive index based on information theory, and we validate its applicability to regulatory scenarios in which the TF binds significantly to the genomic background. Our results show a logarithmic dependence of the evolved information content on the occupancy of target sites and indicate that TFs may actively exploit pseudo-sites to modulate their occupancy of target sites. In regulatory networks with differentially regulated targets, we observe that information content in TF-binding motifs is dictated primarily by the fraction of total probability mass that the TF assigns to its target sites, and we provide a predictive index to estimate the amount of information associated with arbitrarily complex regulatory systems. We observe that complex regulatory patterns can exert additional demands on evolved information content, but, given a total occupancy for target sites, we do not find conclusive evidence that this effect is because of the range of required binding affinities. PMID:24689750

  2. Informational Requirements for Transcriptional Regulation

    PubMed Central

    O'Neill, Patrick K.; Forder, Robert

    2014-01-01

    Abstract Transcription factors (TFs) regulate transcription by binding to specific sites in promoter regions. Information theory provides a useful mathematical framework to analyze the binding motifs associated with TFs but imposes several assumptions that limit their applicability to specific regulatory scenarios. Explicit simulations of the co-evolution of TFs and their binding motifs allow the study of the evolution of regulatory networks with a high degree of realism. In this work we analyze the impact of differential regulatory demands on the information content of TF-binding motifs by means of evolutionary simulations. We generalize a predictive index based on information theory, and we validate its applicability to regulatory scenarios in which the TF binds significantly to the genomic background. Our results show a logarithmic dependence of the evolved information content on the occupancy of target sites and indicate that TFs may actively exploit pseudo-sites to modulate their occupancy of target sites. In regulatory networks with differentially regulated targets, we observe that information content in TF-binding motifs is dictated primarily by the fraction of total probability mass that the TF assigns to its target sites, and we provide a predictive index to estimate the amount of information associated with arbitrarily complex regulatory systems. We observe that complex regulatory patterns can exert additional demands on evolved information content, but, given a total occupancy for target sites, we do not find conclusive evidence that this effect is because of the range of required binding affinities. PMID:24689750

  3. EPA regulations require close study

    SciTech Connect

    Wilkins, J.D.

    1981-08-03

    The time to review environmental legislation and to pinpoint unreasonable or unnecessary sections is when the proposed regulation is published in the Federal Register before it becomes law. Four oil and gas industry organizations can help track and evaluate environmental regulations: the A.G.A.'s Environmental Coordination Group, the Interstate Natural Gas Association of America (INGAA) Environmental Subcommittee, the Gulf Coast Environmental Affairs Group, and the Houston Environmental Roundtable. Besides acting as a watchdog, the industry must perform independent studies to provide valid data for writing the regulations. Once the laws are passed, the industry has no choice but to comply; a plea of ignorance will not change the situation.

  4. Regulations which require employee training: An overview

    SciTech Connect

    Gilfus, L.J.

    1995-11-01

    In order to identify the Occupational Safety and Health Administration (OSHA) regulations which apply to a certain plant or facility, one needs to find the reference materials that are needed and that can be trusted. An overview is presented of the following: Title 29 -- Labor, OSHA Training Requirements; Title 40, Environmental Protection Agency Training Requirements; and Title 49, Department of Transportation Training Regulations.

  5. MAPPING GASOLINE REQUIREMENTS, APPLICABLE REGULATIONS AND BANS

    EPA Science Inventory

    Federal and State regulations play an important role in understanding gasoline composition around the United States. Multiple sources of information on these programs were used to develop reliable, up-to-date maps showing gasoline requirements imposed by various regulations. Th...

  6. 77 FR 51784 - Federal Acquisition Regulation; Information Collection; Qualification Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... minutes per submission assuming an offeror's use of electronic information tracking and retrieval... Regulation; Information Collection; Qualification Requirements AGENCY: Department of Defense (DOD), General... reinstatement request for an information collection requirement regarding an existing OMB clearance....

  7. 77 FR 12754 - Contractor Legal Management Requirements; Acquisition Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-02

    ... Energy Acquisition Regulation (DEAR) (76 FR 81408). The comment period is reopened until March 16, 2012... published a NOPR in the Federal Register (76 FR 81408) to revise existing regulations covering contractor... Part 719 48 Parts 931, 952 and 970 RIN 1990-AA37 Contractor Legal Management Requirements;...

  8. General requirements for RCRA regulated hazardous waste tanks

    SciTech Connect

    1995-11-01

    The Resource Conservation and Recovery Act (RCRA), as amended, requires that tanks used for the storage or treatment of hazardous waste (HazW) be permitted, and comply with the requirements contained within the Code of Federal Regulations (CFR) TItle 40 in Subpart J of Part 264/265, unless those tanks have been exempted. Subpart J specifies requirements for the design, construction, installation, operation, inspection, maintenance, repair, release, response, and closure of HazW tanks. Also, the regulations make a distinction between new and existing tanks. Effective December 6, 1995, standards for controlling volatile organic air emissions will apply to non-exempt HazW tanks. HazW tanks will have to be equipped with a cover or floating roof, or be designed to operate as a closed system, to be in compliance with the air emission control requirements. This information brief describes those tanks that are subject to the Subpart J requirements, and will also discuss secondary containment, inspection, restrictions on waste storage, release response, and closure requirements associated with regulated HazW tanks.

  9. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  10. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  11. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  12. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  13. 40 CFR 63.312 - Existing regulations and requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... National Emission Standards for Coke Oven Batteries § 63.312 Existing regulations and requirements. (a) The..., topside port lids, coke oven doors, and charging operations in effect on September 15, 1992, or which have... method of monitoring in effect on September 15, 1992, and that ensures coke oven emission...

  14. 76 FR 22070 - Federal Acquisition Regulation; Service Contracts Reporting Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-20

    ...DoD, GSA, and NASA are proposing to amend the Federal Acquisition Regulation (FAR) to implement section 743 of Division C of the Consolidated Appropriations Act, 2010. This proposed rule amends the FAR to require service contractors for executive agencies, except the Department of Defense (DoD), covered by the Federal Activities Inventory Reform (FAIR) Act of 1998, to submit information......

  15. 78 FR 80369 - Federal Acquisition Regulation; Service Contracts Reporting Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-31

    ...DoD, GSA, and NASA are issuing a final rule amending the Federal Acquisition Regulation (FAR) to implement a section of the Consolidated Appropriations Act, 2010. This final rule amends the FAR to require service contractors for executive agencies, except where DoD has fully funded the contract or order, to submit information annually in support of agency-level inventories for service......

  16. Review of orders and regulations requiring environmental protection

    SciTech Connect

    Kelly, E.; Cunningham, R.; Michael, D.

    1996-09-01

    With the increased awareness of and interest in potential ecological risks associated with past, current, and future Department of Energy (DOE) activities, DOE`s Defense Programs (DP) Office of Technical and Environmental Support sponsored a study to (1) evaluate the effectiveness of the current compliance-driven environmental protection and assessment efforts relative to ecological concerns; (2) explore the need for a more focused, integrated approach to address ecological impacts; and (3) identify the requirements for an integrated approach. The study explored four questions. (a) Which federal regulations and DOE orders either explicitly require ecological assessments or implicitly require them through environmental protection language? (b) What currently is being done at selected DOE facilities to implement these regulations and orders? (c) What are private sector industries doing in terms of ecological risk assessments and how do industry approaches and issues compare with those of DOE? (d) What, if anything, in addition to current efforts is needed to ensure the protection of ecological resources associated with DOE facilities, to support defensible decision making, and to improve efficiency? The results of this study are presented in a report titled {open_quotes}Integrated, Comprehensive Ecological Impact Assessments In Support of Department of Energy Decision Making{close_quotes}. This report is a companion document to that report. This report provides a more detailed discussion of the document reviews of the relevant environmental protection regulations and current and pending DOE orders. The main goal of the document reviews was to understand existing requirements for ecological data collection and impact assessments.

  17. Actin is required for IFT regulation in Chlamydomonas reinhardtii

    PubMed Central

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C.; Sale, Winfield S.; Shoichet, Brian; Pringle, John R.; Marshall, Wallace F.

    2014-01-01

    Summary Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Since actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here, we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation, and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor suggesting actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

  18. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    PubMed

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

  19. 75 FR 6186 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 205...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-08

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 205, Publicizing Contract Actions AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  20. 75 FR 12518 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 237...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-16

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 237, Service Contracting AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  1. 78 FR 15935 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-13

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS); Bonds and Insurance AGENCY: Defense Acquisition Regulations System, Department... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  2. 76 FR 50461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Types of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-15

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Types of Contracts AGENCY: Defense Acquisition Regulations System, Department of... information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of the...

  3. 75 FR 20825 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Pilot...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Pilot Mentor-Protege Program AGENCY: Defense Acquisition Regulations System... approved information collection requirement. ] SUMMARY: In compliance with section 3506(c)(2)(A) of...

  4. 75 FR 20825 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 211...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-21

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 211, Describing Agency Needs AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  5. 78 FR 70025 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-22

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Subcontracting Policies and Procedures AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with section 3506(c)(2)(A) of...

  6. 77 FR 30998 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Government...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-24

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Government Property AGENCY: Defense Acquisition Regulations System, Department of... and Technical Information System,'' and is used when directed by the plant clearance officer. e....

  7. 77 FR 43076 - Federal Acquisition Regulation; Information Collection; Value Engineering Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-23

    ... Regulation; Information Collection; Value Engineering Requirements AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Value Engineering Requirements. Public comments are...: Submit comments identified by Information Collection 9000- 0027, Value Engineering Requirements, by...

  8. 75 FR 45104 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments. SUMMARY: In... announces the proposed extension of a public information collection requirement and seeks public comment...

  9. 75 FR 68331 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DFARS Part...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed extension of an approved information collection requirement. SUMMARY: In compliance...

  10. 75 FR 26739 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 244...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition...: Defense Acquisition Regulations System, Department of Defense. ACTION: Notice and request for comments regarding a proposed extension of an approved information collection requirement. SUMMARY: In...

  11. 78 FR 17186 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-20

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed extension of an approved information collection requirement. SUMMARY: In compliance...

  12. 75 FR 10790 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-09

    ... Regulation Supplement; Notification Requirements for Critical Safety Items AGENCY: Defense Acquisition... Supplement (DFARS) Notification Requirements for Critical Safety Items; OMB Control Number 0704-0441....

  13. Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions

    PubMed Central

    Mittag, Jens; Lyons, David J.; Sällström, Johan; Vujovic, Milica; Dudazy-Gralla, Susi; Warner, Amy; Wallis, Karin; Alkemade, Anneke; Nordström, Kristina; Monyer, Hannah; Broberger, Christian; Arner, Anders; Vennström, Björn

    2012-01-01

    Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone’s central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1. PMID:23257356

  14. translin Is Required for Metabolic Regulation of Sleep.

    PubMed

    Murakami, Kazuma; Yurgel, Maria E; Stahl, Bethany A; Masek, Pavel; Mehta, Aradhana; Heidker, Rebecca; Bollinger, Wesley; Gingras, Robert M; Kim, Young-Joon; Ja, William W; Suter, Beat; DiAngelo, Justin R; Keene, Alex C

    2016-04-01

    Dysregulation of sleep or feeding has enormous health consequences. In humans, acute sleep loss is associated with increased appetite and insulin insensitivity, while chronically sleep-deprived individuals are more likely to develop obesity, metabolic syndrome, type II diabetes, and cardiovascular disease. Conversely, metabolic state potently modulates sleep and circadian behavior; yet, the molecular basis for sleep-metabolism interactions remains poorly understood. Here, we describe the identification of translin (trsn), a highly conserved RNA/DNA binding protein, as essential for starvation-induced sleep suppression. Strikingly, trsn does not appear to regulate energy stores, free glucose levels, or feeding behavior suggesting the sleep phenotype of trsn mutant flies is not a consequence of general metabolic dysfunction or blunted response to starvation. While broadly expressed in all neurons, trsn is transcriptionally upregulated in the heads of flies in response to starvation. Spatially restricted rescue or targeted knockdown localizes trsn function to neurons that produce the tachykinin family neuropeptide Leucokinin. Manipulation of neural activity in Leucokinin neurons revealed these neurons to be required for starvation-induced sleep suppression. Taken together, these findings establish trsn as an essential integrator of sleep and metabolic state, with implications for understanding the neural mechanism underlying sleep disruption in response to environmental perturbation. PMID:27020744

  15. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    SciTech Connect

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  16. 77 FR 67366 - Federal Acquisition Regulation; Information Collection; Quality Assurance Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-09

    ... Regulation; Information Collection; Quality Assurance Requirements AGENCY: Department of Defense (DOD... information collection requirement concerning quality assurance requirements. Public comments are particularly... and methodology; ways to enhance the quality, utility, and clarity of the information to be...

  17. 78 FR 19467 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-01

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS); DoD Pilot Mentor-Prot g Program AGENCY: Defense Acquisition Regulations System... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  18. 78 FR 60898 - Regulation on Definition and Requirements for a Nationally Recognized Testing Laboratory...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-02

    ...OSHA requests comments concerning its proposed revision and extension of the information collection requirements specified by its Regulation at 29 CFR 1910.7, ``definition and requirements for a nationally recognized testing laboratory'' (The Regulation). The Regulation specifies procedures that organizations must follow to apply for, and to maintain, OSHA's recognition to test and certify......

  19. 76 FR 72914 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Government...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Foreign Disclosure and Technical Information System,'' and is used when directed by the plant clearance... Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments. SUMMARY:...

  20. 75 FR 68333 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition...-0248) AGENCY: Defense Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed extension of an approved information collection...

  1. 75 FR 41161 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Part 251...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-15

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... vehicles under the Interagency Fleet Management System (IFMS) and obtain related services. The information... Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a...

  2. 76 FR 35424 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-17

    ... Regulation Supplement; Acquisition of Information Technology AGENCY: Defense Acquisition Regulations System... technology. The Office of Management and Budget (OMB) has approved this information collection requirement... Supplement (DFARS) Part 239, Acquisition of Information Technology, and the associated clauses at DFARS...

  3. 78 FR 63462 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-24

    ... organizational conflicts of interest arising in a systems engineering and technical assistance contract for an... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition...: Defense Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request...

  4. 78 FR 25795 - Contractor Legal Management Requirements; Acquisition Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-03

    ... Federal Register on August 31, 1994 (59 FR 44981). The interim Acquisition Letter was finalized as a Policy Statement on April 3, 1996 (61 FR 14763). This Policy Statement was followed by a formal... Regulations with an effective date of April 23, 2001 (66 FR 4616, 66 FR 19717). After a decade operating...

  5. Cholesterol and regulated exocytosis: a requirement for unitary exocytotic events.

    PubMed

    Rituper, Boštjan; Flašker, Ajda; Guček, Alenka; Chowdhury, Helena H; Zorec, Robert

    2012-01-01

    Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes. PMID:22726879

  6. 78 FR 68831 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-15

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed extension of an approved information collection requirement. SUMMARY: In compliance with Section...

  7. 78 FR 67133 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-08

    ... holders further disseminate the information to other firms within a geographic area defined in the... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... approved information collection requirement. SUMMARY: In compliance with Section 3506(c)(2)(A) of...

  8. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Motor carrier to require observance of driver regulations. 390.11 Section 390.11 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION FEDERAL MOTOR CARRIER SAFETY REGULATIONS FEDERAL MOTOR CARRIER...

  9. 49 CFR 379.11 - Waiver of requirements of the regulations in this part.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Waiver of requirements of the regulations in this part. 379.11 Section 379.11 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL MOTOR CARRIER SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION FEDERAL MOTOR CARRIER SAFETY REGULATIONS PRESERVATION OF RECORDS §...

  10. 7 CFR 318.13-3 - General requirements for all regulated articles.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 5 2011-01-01 2011-01-01 false General requirements for all regulated articles. 318.13-3 Section 318.13-3 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE STATE OF HAWAII AND TERRITORIES QUARANTINE NOTICES Regulated Articles From Hawaii and...

  11. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 false What is a regulated rural gathering line and what...) PIPELINE SAFETY TRANSPORTATION OF HAZARDOUS LIQUIDS BY PIPELINE General § 195.11 What is a regulated rural gathering line and what requirements apply? Each operator of a regulated rural gathering line, as defined...

  12. 77 FR 24611 - Removal of Regulations Requiring 3% Withholding by Government Entities

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-25

    ... 9524, 76 FR 26583, 2011-23 IRB 843) (the May 2011 final regulations). Those regulations were issued to... in the Federal Register on May 9, 2011 (REG-151687-10, 76 FR 26678, 2011-23 IRB 867). A related... Internal Revenue Service 26 CFR Part 31 RIN 1545-BK83 Removal of Regulations Requiring 3% Withholding...

  13. 76 FR 35218 - Federal Acquisition Regulation; Information Collection; Cost or Pricing Data Requirements and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-16

    ... Regulation; Information Collection; Cost or Pricing Data Requirements and Information Other Than Cost or... requirement concerning cost or pricing data requirements and information other than cost or pricing data... Information Collection 9000- 0013, Cost or Pricing Data Requirements and Information Other Than Cost...

  14. 77 FR 29983 - Federal Acquisition Regulation; Information Collection; Novation/Change of Name Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-21

    ... Regulation; Information Collection; Novation/ Change of Name Requirements AGENCY: Department of Defense (DOD... collection requirement concerning Novation/Change of Name Requirements. Public comments are particularly... comments identified by Information Collection 9000- 0076, Novation/Change of Name Requirements, by any...

  15. 76 FR 71558 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Requests...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-18

    ... Defense Acquisition Regulations System Information Collection Requirement; Defense Federal Acquisition... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments. SUMMARY...), DoD announces the proposed extension of a public information collection requirement and seeks...

  16. 49 CFR 1220.5 - Waiver of requirements of these regulations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 9 2010-10-01 2010-10-01 false Waiver of requirements of these regulations. 1220.5 Section 1220.5 Transportation Other Regulations Relating to Transportation (Continued) SURFACE TRANSPORTATION BOARD, DEPARTMENT OF TRANSPORTATION (CONTINUED) ACCOUNTS, RECORDS AND REPORTS PRESERVATION OF RECORDS § 1220.5 Waiver of requirements...

  17. 76 FR 71707 - Revising Underground Storage Tank Regulations-Revisions to Existing Requirements and New...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-18

    ...EPA is proposing to make certain revisions to the 1988 underground storage tank (UST) technical, financial responsibility, and state program approval regulations. These changes establish federal requirements that are similar to key portions of the Energy Policy Act of 2005; they also update certain 1988 UST regulations. Proposed changes include: Adding secondary containment requirements for......

  18. 75 FR 60263 - Federal Acquisition Regulation; Offering a Construction Requirement-8(a) Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-29

    ... Federal Acquisition Regulation; Offering a Construction Requirement--8(a) Program AGENCIES: Department of... submitting offering letters to SBA for a construction requirement for which a specific offeror is nominated... offerings for construction ] requirements be submitted to the SBA District Office for the geographical...

  19. 78 FR 25445 - Federal Acquisition Regulation; Submission for OMB Review; Quality Assurance Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-01

    ... information collection requirement concerning quality assurance requirements. A notice was published in the Federal Register at 77 FR 67366, on November 11, 2012. No comments were received. DATES: Submit comments... Regulation; Submission for OMB Review; Quality Assurance Requirements AGENCY: Department of Defense...

  20. 77 FR 62495 - Federal Acquisition Regulation; Submission for OMB Review; Novation/Change of Name Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-15

    ... approved information collection requirement concerning Novation/Change of Name Requirements. A notice was published in the Federal Register at 77 FR 29983, on May 21, 2012. One respondent submitted comments. Public... Regulation; Submission for OMB Review; Novation/Change of Name Requirements AGENCY: Department of...

  1. 12 CFR 749.5 - Format for records required by other NCUA regulations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Format for records required by other NCUA regulations. 749.5 Section 749.5 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING CREDIT UNIONS RECORDS PRESERVATION PROGRAM AND APPENDICES-RECORD RETENTION GUIDELINES; CATASTROPHIC...

  2. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  3. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  4. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  5. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... operation of distilled spirits plants. Part 19 of title 27 CFR contains the regulations relating to the...) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous regulations relative to the... requirements therefor. (3) (4) Gauging of distilled spirits. Part 30 of title 27 CFR contains the...

  6. 78 FR 72620 - Federal Acquisition Regulation; Higher-Level Contract Quality Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-03

    ... comment in the Federal Register at 78 FR 28780 on May 16, 2013. 2. FAR Case 2013-002, entitled ``Expanded... RIN 9000-AM65 Federal Acquisition Regulation; Higher-Level Contract Quality Requirements AGENCY... Acquisition Regulation (FAR) to clarify when to use higher-level quality standards in solicitations...

  7. 12 CFR 350.12 - Disclosure required by applicable banking or securities law or regulations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 4 2011-01-01 2011-01-01 false Disclosure required by applicable banking or securities law or regulations. 350.12 Section 350.12 Banks and Banking FEDERAL DEPOSIT INSURANCE CORPORATION... STATE NONMEMBER BANKS § 350.12 Disclosure required by applicable banking or securities law...

  8. 75 FR 14448 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-25

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... announcing a public workshop entitled ``FDA Clinical Trial Requirements, Regulations, Compliance, and Good... representatives. The program will focus on the relationships among the FDA and clinical trial staff,...

  9. 77 FR 59139 - Prompt Corrective Action, Requirements for Insurance, and Promulgation of NCUA Rules and Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-26

    ... amended by IRPS 03-2, and two NCUA regulations that apply asset thresholds to grant relief from risk-based...'' credit union for determining whether risk-based net worth requirements apply, and 12 CFR 741.3(b)(5)(i... interest rate risk rule requirements. \\1\\ IRPS 03-2, 68 FR 31949 (May 29, 2003). B. Why is the...

  10. 76 FR 72915 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; DoD Acquisition Process (Various Miscellaneous Requirements) (OMB Control Number 0704-0187) AGENCY: Department of Defense (DoD... Paperwork Reduction Act of 1995 (44 U.S.C. chapter 35), DoD announces the proposed extension of a...

  11. 12 CFR 350.12 - Disclosure required by applicable banking or securities law or regulations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 4 2010-01-01 2010-01-01 false Disclosure required by applicable banking or securities law or regulations. 350.12 Section 350.12 Banks and Banking FEDERAL DEPOSIT INSURANCE CORPORATION... STATE NONMEMBER BANKS § 350.12 Disclosure required by applicable banking or securities law...

  12. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  13. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 5 2012-10-01 2012-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  14. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 5 2013-10-01 2013-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  15. 49 CFR 390.11 - Motor carrier to require observance of driver regulations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 5 2014-10-01 2014-10-01 false Motor carrier to require observance of driver... Motor carrier to require observance of driver regulations. Whenever in part 325 of subchapter A or in this subchapter a duty is prescribed for a driver or a prohibition is imposed upon the driver, it...

  16. 78 FR 4725 - Escrow Requirements Under the Truth in Lending Act (Regulation Z)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-22

    ...The Bureau of Consumer Financial Protection (Bureau) is publishing a final rule that amends Regulation Z (Truth in Lending) to implement certain amendments to the Truth in Lending Act made by the Dodd-Frank Wall Street Reform and Consumer Protection Act (Dodd-Frank Act). Regulation Z currently requires creditors to establish escrow accounts for higher-priced mortgage loans secured by a first......

  17. Pre-LTP requires extracellular signal-regulated kinase in the ACC

    PubMed Central

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush

    2016-01-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  18. Pre-LTP requires extracellular signal-regulated kinase in the ACC.

    PubMed

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush; Zhuo, Min

    2016-02-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  19. 78 FR 4788 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-23

    ...The Commander, Ninth Coast Guard District is staying (suspending) reporting requirements under the Regulated Navigation Area (RNA) established for barges loaded with certain dangerous cargoes (CDC barges) in the inland rivers of the Ninth Coast Guard District. This stay (suspension) extension is necessary because the Coast Guard continues to analyze future reporting needs and evaluate possible......

  20. 78 FR 61183 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-03

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA11 Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous Cargoes, Inland Rivers, Ninth Coast Guard District; Stay (Suspension) AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Commander, Ninth Coast...

  1. 76 FR 1360 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-10

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA11 Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous Cargoes, Inland Rivers, Eighth Coast Guard District; Stay (Suspension) AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Commander, Eighth...

  2. 21 CFR 16.24 - Regulatory hearing required by the act or a regulation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Regulatory hearing required by the act or a regulation. 16.24 Section 16.24 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL REGULATORY HEARING BEFORE THE FOOD AND DRUG ADMINISTRATION Initiation of Proceedings §...

  3. 2 CFR 25.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 2 Grants and Agreements 1 2012-01-01 2012-01-01 false Requirements for program announcements, regulations, and application instructions. 25.200 Section 25.200 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS...

  4. 78 FR 73104 - Federal Travel Regulation (FTR); Agency Requirements for Payment of Expenses Connected With the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-05

    ... Heroes Act,'' dated September 12, 2011, and published in the Federal Register (76 FR 57621), delegates to...] RIN 3090-AJ37 Federal Travel Regulation (FTR); Agency Requirements for Payment of Expenses Connected.... General Services Administration (GSA). ACTION: Final rule. ] SUMMARY: GSA is amending the Federal...

  5. 26 CFR 56.6001-1 - Notice or regulations requiring records, statements, and special returns.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 17 2011-04-01 2011-04-01 false Notice or regulations requiring records, statements, and special returns. 56.6001-1 Section 56.6001-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) PUBLIC CHARITY EXCISE...

  6. 26 CFR 56.6001-1 - Notice or regulations requiring records, statements, and special returns.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 17 2010-04-01 2010-04-01 false Notice or regulations requiring records, statements, and special returns. 56.6001-1 Section 56.6001-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) PUBLIC CHARITY EXCISE...

  7. 13 CFR 120.1511 - Certification and other reporting and notification requirements for Other Regulated SBLCs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Certification and other reporting and notification requirements for Other Regulated SBLCs. 120.1511 Section 120.1511 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION BUSINESS LOANS Risk-Based Lender Oversight Enforcement Actions §...

  8. 50 CFR 14.254 - What are the requirements contained in these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 1 2010-10-01 2010-10-01 false What are the requirements contained in these regulations? 14.254 Section 14.254 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR TAKING, POSSESSION, TRANSPORTATION, SALE, PURCHASE, BARTER, EXPORTATION, AND IMPORTATION OF WILDLIFE AND...

  9. 9 CFR 201.4 - Bylaws, rules and regulations, and requirements of exchanges, associations, or other...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Bylaws, rules and regulations, and requirements of exchanges, associations, or other organizations; applicability, establishment. 201.4 Section 201.4 Animals and Animal Products GRAIN INSPECTION, PACKERS AND STOCKYARDS ADMINISTRATION (PACKERS...

  10. 78 FR 11279 - Loan Originator Compensation Requirements Under the Truth in Lending Act (Regulation Z)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-15

    ...The Bureau of Consumer Financial Protection (Bureau) is amending Regulation Z to implement amendments to the Truth in Lending Act (TILA) made by the Dodd-Frank Wall Street Reform and Consumer Protection Act (Dodd-Frank Act). The final rule implements requirements and restrictions imposed by the Dodd-Frank Act concerning loan originator compensation; qualifications of, and registration or......

  11. 2 CFR 25.200 - Requirements for program announcements, regulations, and application instructions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 2 Grants and Agreements 1 2014-01-01 2014-01-01 false Requirements for program announcements, regulations, and application instructions. 25.200 Section 25.200 Grants and Agreements Office of Management and Budget Guidance for Grants and Agreements OFFICE OF MANAGEMENT AND BUDGET GOVERNMENTWIDE GUIDANCE FOR GRANTS AND AGREEMENTS Reserved...

  12. 50 CFR 25.23 - What are the general regulations and information collection requirements?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 8 2011-10-01 2011-10-01 false What are the general regulations and information collection requirements? 25.23 Section 25.23 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR (CONTINUED) THE NATIONAL WILDLIFE REFUGE SYSTEM ADMINISTRATIVE PROVISIONS Administrative Provisions §...

  13. 50 CFR 25.23 - What are the general regulations and information collection requirements?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false What are the general regulations and information collection requirements? 25.23 Section 25.23 Wildlife and Fisheries UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR (CONTINUED) THE NATIONAL WILDLIFE REFUGE SYSTEM ADMINISTRATIVE PROVISIONS Administrative Provisions §...

  14. 75 FR 29456 - Acquisition Regulation: Subchapter E-General Contracting Requirements, Subchapter F-Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-26

    ...The Department of Energy (DOE) is amending the Department of Energy Acquisition Regulation (DEAR) Subchapters E--General Contracting Requirements, F--Special Categories of Contracting, and G--Contract Management to make changes to conform to the FAR, remove out-of-date coverage, and to update references. Today's rule does not alter substantive rights or obligations under current...

  15. 75 FR 964 - Acquisition Regulation: Subchapter E-General Contracting Requirements, Subchapter F-Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-07

    ...The Department of Energy (DOE) is proposing to amend the Department of Energy Acquisition Regulation (DEAR) Subchapters E-- General Contracting Requirements, F--Special Categories of Contracting, and G--Contract Management to make changes to conform to the FAR, remove out-of-date coverage, and to update references. DOE will separately propose rules for changes to parts 927 and 945,......

  16. Medical device data systems and FDA regulation. Should medical device data systems require FDA clearance?

    PubMed

    Kelley, Peter

    2010-01-01

    It is widely understood why medical devices need to be regulated by the FDA and other governing bodies. However medical software does not typically require the same level of regulation. Currently the FDA is investigating whether one type of medical software, Medical Device Data Systems (MDDS), should require FDA clearance because of the potential risk they impose when interconnected with medical devices. Hospitals are looking to implement MDDS because the technology allows nursing staff to spend more time on direct patient care and reduces charting errors. This article will explore the FDA's proposal and will review the possible risks and provide a rationale for why MDDS should be regulated by the FDA and why MDDS vendors should have the right level of quality and risk management procedures in place to ensure that they are developing and bringing to market the safest products possible. PMID:20677470

  17. Additional requirements for charitable hospitals; community health needs assessments for charitable hospitals; requirement of a section 4959 excise tax return and time for filing the return. Final regulations and removal of temporary regulations.

    PubMed

    2014-12-31

    This document contains final regulations that provide guidance regarding the requirements for charitable hospital organizations added by the Patient Protection and Affordable Care Act of 2010. The regulations will affect charitable hospital organizations PMID:25562896

  18. RCRA corrective action permit requirements and modifications under Subpart F regulations. RCRA Information Brief

    SciTech Connect

    Coalgate, J.

    1993-07-01

    The ground water protection requirements under the Resource Conservation and Recovery Act (RCRA), 40 CFR 264, Subpart F, apply to surface impoundments, waste plies, land treatment units, and landfills that received hazardous waste after July 26,1982 (i.e., regulated units). There are three phases to the Subpart F ground water protection requirements: detection monitoring, compliance monitoring, and corrective action. Subpart F corrective action applies to remediation of ground water contamination resulting from releases from regulated units at a treatment, storage, or disposal facility (TSDF). The TSDF owner or operator is responsible for complying with these requirements. This Information Brief provides information on the permit requirements under Subpart F. This Information Brief is one of a series on RCRA corrective action. The first step in the permitting process is for the facility to determine the need for ground-water monitoring. The regulations found in 40 CFR 264 Sections 264.90 to 264.100 (Subpart F) apply to all regulated units. A ``regulated unit`` is defined as a surface impoundment, waste pile, landfill, or land treatment unit that received hazardous waste after July 26, 1982. Such units require a permit under RCRA. Subpart F entails a three-phased program designed to detect, evaluate, and, if necessary, respond to ground water contamination. The ground-water protection standard, including identification of maximum contaminant levels (MCLs) under the Safe Drinking Water Act (SDWA) and alternate concentration limits (ACLs), is established with the permit application. Where MCLs and ACLs cannot be established, the standard may be established at background levels.

  19. Root growth regulation and gravitropism in maize roots does not require the epidermis

    NASA Technical Reports Server (NTRS)

    Bjorkman, T.; Cleland, R. E.

    1991-01-01

    We have earlier published observations showing that endogenous alterations in growth rate during gravitropism in maize roots (Zea mays L.) are unaffected by the orientation of cuts which remove epidermal and cortical tissue in the growing zone (Bjorkman and Cleland, 1988, Planta 176, 513-518). We concluded that the epidermis and cortex are not essential for transporting a growth-regulating signal in gravitropism or straight growth, nor for regulating the rate of tissue expansion. This conclusion has been challenged by Yang et al. (1990, Planta 180, 530-536), who contend that a shallow girdle around the entire perimeter of the root blocks gravitropic curvature and that this inhibition is the result of a requirement for epidermal cells to transport the growth-regulating signal. In this paper we demonstrate that the entire epidermis can be removed without blocking gravitropic curvature and show that the position of narrow girdles does not affect the location of curvature. We therefore conclude that the epidermis is not required for transport of a growth-regulating substance from the root cap to the growing zone, nor does it regulate the growth rate of the elongating zone of roots.

  20. Characterization of parameters required for effective use of tamoxifen-regulated recombination.

    PubMed

    Buelow, Ben; Scharenberg, Andrew M

    2008-01-01

    Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population. PMID:18810268

  1. An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

    PubMed Central

    Hecht, G B; Lane, T; Ohta, N; Sommer, J M; Newton, A

    1995-01-01

    Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints. Images PMID:7664732

  2. p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network.

    PubMed

    Marshall, Clayton B; Mays, Deborah J; Beeler, J Scott; Rosenbluth, Jennifer M; Boyd, Kelli L; Santos Guasch, Gabriela L; Shaver, Timothy M; Tang, Lucy J; Liu, Qi; Shyr, Yu; Venters, Bryan J; Magnuson, Mark A; Pietenpol, Jennifer A

    2016-03-15

    We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues. PMID:26947080

  3. p73 is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network

    PubMed Central

    Marshall, Clayton B; Mays, Deborah J; Beeler, J Scott; Rosenbluth, Jennifer M; Boyd, Kelli L; Guasch, Gabriela L Santos; Shaver, Timothy M; Tang, Lucy J; Liu, Qi; Shyr, Yu; Venters, Bryan J; Magnuson, Mark A; Pietenpol, Jennifer A

    2016-01-01

    Summary We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus, hippocampal dysgenesis, sterility and chronic inflammation/infection of lung, middle ear and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells, and suggest that p73 ‘marks’ these cells for MCC differentiation. In sum, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation and, like p63, has an essential role in development of tissues. PMID:26947080

  4. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  5. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  6. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... regulations contained in 33 CFR 165.13 apply to this section. (e) Eighth Coast Guard District Inland River RNA...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415...

  7. The Global Regulator CodY Regulates Toxin Gene Expression in Bacillus anthracis and Is Required for Full Virulence▿ †

    PubMed Central

    van Schaik, Willem; Château, Alice; Dillies, Marie-Agnès; Coppée, Jean-Yves; Sonenshein, Abraham L.; Fouet, Agnès

    2009-01-01

    In gram-positive bacteria, CodY is an important regulator of genes whose expression changes upon nutrient limitation and acts as a repressor of virulence gene expression in some pathogenic species. Here, we report the role of CodY in Bacillus anthracis, the etiologic agent of anthrax. Disruption of codY completely abolished virulence in a toxinogenic, noncapsulated strain, indicating that the activity of CodY is required for full virulence of B. anthracis. Global transcriptome analysis of a codY mutant and the parental strain revealed extensive differences. These differences could reflect direct control for some genes, as suggested by the presence of CodY binding sequences in their promoter regions, or indirect effects via the CodY-dependent control of other regulatory proteins or metabolic rearrangements in the codY mutant strain. The differences included reduced expression of the anthrax toxin genes in the mutant strain, which was confirmed by lacZ reporter fusions and immunoblotting. The accumulation of the global virulence regulator AtxA protein was strongly reduced in the mutant strain. However, in agreement with the microarray data, expression of atxA, as measured using an atxA-lacZ transcriptional fusion and by assaying atxA mRNA, was not significantly affected in the codY mutant. An atxA-lacZ translational fusion was also unaffected. Overexpression of atxA restored toxin component synthesis in the codY mutant strain. These results suggest that CodY controls toxin gene expression by regulating AtxA accumulation posttranslationally. PMID:19651859

  8. Using Five-Minute Data to Allocate Load-Following and Regulation Requirements among Individual Customers

    SciTech Connect

    Kirby, B.J.

    2001-03-27

    B. Kirby and E. Hirst, Customer-Specific Metrics for the Regulation and Load-Following 1 Ancillary Services, ORNL/CON-474, Oak Ridge National Laboratory, Oak Ridge, Tenn., January 2000. In an earlier project, we analyzed data on total system load and the loads of eight large industrial customers in terms of system- and customer-specific requirements for the regulation and load-following ancillary services. We conducted these analyses using 12 days of data from February 1 1999 plus 12 days of data from August and September 1999. These analyses were conducted using data provided by the control area at the 30-s level, which we then aggregated to the 2-min level for subsequent analysis. The current project analyzes the feasibility of using 5-min revenue-meter (RM) data to allocate load-following and regulation requirements among retail customers. This project does not use the 5-min data to determine the actual ancillary services requirements for individual loads because these requirements depend strongly on the time-averaging period chosen for the load data. In particular, the amount of regulation required declines as the time-averaging period increases. Our earlier project showed that 2 min was a reasonable time-averaging period for this control area. The reason for examining 5-min data is that supervisory control and data acquisition (SCADA) data, which are available every 4 s, are collected for only eight of this control area's very largest industrial customers. On the other hand, this control area collects 5-min RM data for about 600 customers. Although these customers account for less than 0.2% of the retail customers, they account for 60% of total energy sales and more than 40% of total revenues. Thus, if the allocations obtained with 5-min data for these eight large customers agree well with the allocations obtained with the SCADA data for these customers, the method we developed to allocate regulation costs to individual customers can be applied to many more

  9. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

    PubMed Central

    Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

    2013-01-01

    Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

  10. TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

    PubMed Central

    Shambharkar, Prashant B.; Bittinger, Mark; Latario, Brian; Xiong, ZhaoHui; Bandyopadhyay, Somnath; Davis, Vanessa; Lin, Victor; Yang, Yi; Valdez, Reginald; Labow, Mark A.

    2015-01-01

    Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis. PMID:25996873

  11. IL-12 is required for mTOR regulation of memory CTLs during viral infection

    PubMed Central

    Garcia, Karla; Sun, Zhifeng; Mattson, Elliot; Li, Lei; Smyth, Kendra; Xiao, Zhengguo

    2014-01-01

    The induction of functional memory CTLs is a major goal of vaccination against intracellular pathogens. IL-12 is critical for the generation of memory CTLs, and inhibition of mTOR by rapamycin can effectively enhance the memory CTL response. Yet, the role of IL-12 in mTOR’s regulation of memory CTL is unknown. Here, we hypothesized that the immunostimulatory effects of mTOR on memory CTLs requires IL-12 signaling. Our results revealed that rapamycin increased the generation of memory CTLs in vaccinia virus infection, and this enhancement was dependent upon the IL-12 signal. Furthermore, IL-12 receptor deficiency diminished the secondary expansion of rapamycin-regulated memory, and resultant secondary memory CTLs were abolished. Rapamycin enhanced IL-12 signaling by up regulating IL-12 receptor β2 expression and STAT4 phosphorylation in CTLs during early infection. In addition, rapamycin continually suppressed T-bet expression in both WT and IL-12 receptor knockout CTLs. These results indicate an essential role for IL-12 in the regulation of memory CTLs by mTOR, and highlight the importance of considering the interplay between cytokines and adjuvants during vaccine design. PMID:24898389

  12. Neto2 is a KCC2 interacting protein required for neuronal Cl− regulation in hippocampal neurons

    PubMed Central

    Ivakine, Evgueni A.; Acton, Brooke A.; Mahadevan, Vivek; Ormond, Jake; Tang, Man; Pressey, Jessica C.; Huang, Michelle Y.; Ng, David; Delpire, Eric; Salter, Michael W.; Woodin, Melanie A.; McInnes, Roderick R.

    2013-01-01

    KCC2 is a neuron-specific K+–Cl− cotransporter that is essential for Cl− homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl− extrusion, resulting in decreased synaptic inhibition in hippocampal neurons. PMID:23401525

  13. Smad4 is required to regulate the fate of cranial neural crest cells

    PubMed Central

    Ko, Seung O; Chung, Il Hyuk; Xu, Xun; Oka, Shoji; Zhao, Hu; Cho, Eui Sic; Deng, Chuxia; Chai, Yang

    2009-01-01

    Smad4 is the central mediator for TGF-β/BMP signals, which are involved in regulating cranial neural crest (CNC) cell formation, migration, proliferation and fate determination. It is unclear whether TGF-β/BMP signals utilize Smad-dependent or –independent pathways to control the development of CNC cells. To investigate the functional significance of Smad4 in regulating CNC cells, we generated mice with neural crest specific inactivation of the Smad4 gene. Our study shows that Smad4 is not required for the migration of CNC cells, but is required in neural crest cells for the development of the cardiac outflow tract. Smad4 is essential in mediating BMP signaling in the CNC-derived ectomesenchyme during early stages of tooth development because conditional inactivation of Smad4 in neural crest derived cells results in incisor and molar development arrested at the dental lamina stage. Furthermore, Smad-mediated TGF-β/BMP signaling controls the homeobox gene patterning of oral/aboral and proximal/distal domains within the first branchial arch. At the cellular level, a Smad4-mediated downstream target gene(s) is required for the survival of CNC cells in the proximal domain of the first branchial arch. Smad4 mutant mice show underdevelopment of the first branchial arch and midline fusion defects. Taken together, our data show that TGF-β/BMP signals rely on Smad-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control craniofacial organogenesis. PMID:17964566

  14. FOXO signaling is required for disuse muscle atrophy and is directly regulated by Hsp70

    PubMed Central

    Senf, Sarah M.; Dodd, Stephen L.

    2010-01-01

    The purpose of the current study was to determine whether heat shock protein 70 (Hsp70) directly regulates forkhead box O (FOXO) signaling in skeletal muscle. This aim stems from previous work demonstrating that Hsp70 overexpression inhibits disuse-induced FOXO transactivation and prevents muscle fiber atrophy. However, although FOXO is sufficient to cause muscle wasting, no data currently exist on the requirement of FOXO signaling in the progression of physiological muscle wasting, in vivo. In the current study we show that specific inhibition of FOXO, via expression of a dominant-negative FOXO3a, in rat soleus muscle during disuse prevented >40% of muscle fiber atrophy, demonstrating that FOXO signaling is required for disuse muscle atrophy. Subsequent experiments determined whether Hsp70 directly regulates FOXO3a signaling when independently activated in skeletal muscle, via transfection of FOXO3a. We show that Hsp70 inhibits FOXO3a-dependent transcription in a gene-specific manner. Specifically, Hsp70 inhibited FOXO3a-induced promoter activation of atrogin-1, but not MuRF1. Further studies showed that a FOXO3a DNA-binding mutant can activate MuRF1, but not atrogin-1, suggesting that FOXO3a activates these two genes through differential mechanisms. In summary, FOXO signaling is required for physiological muscle atrophy and is directly inhibited by Hsp70. PMID:19864323

  15. Unusually long-lived pause required for regulation of a Rho-dependent transcription terminator

    PubMed Central

    Hollands, Kerry; Sevostiyanova, Anastasia; Groisman, Eduardo A.

    2014-01-01

    Up to half of all transcription termination events in bacteria rely on the RNA-dependent helicase Rho. However, the nucleic acid sequences that promote Rho-dependent termination remain poorly characterized. Defining the molecular determinants that confer Rho-dependent termination is especially important for understanding how such terminators can be regulated in response to specific signals. Here, we identify an extraordinarily long-lived pause at the site where Rho terminates transcription in the 5′-leader region of the Mg2+ transporter gene mgtA in Salmonella enterica. We dissect the sequence elements required for prolonged pausing in the mgtA leader and establish that the remarkable longevity of this pause is required for a riboswitch to stimulate Rho-dependent termination in the mgtA leader region in response to Mg2+ availability. Unlike Rho-dependent terminators described previously, where termination occurs at multiple pause sites, there is a single site of transcription termination directed by Rho in the mgtA leader. Our data suggest that Rho-dependent termination events that are subject to regulation may require elements distinct from those operating at constitutive Rho-dependent terminators. PMID:24778260

  16. 75 FR 45104 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-02

    ... Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department of... Federal Acquisition Regulation Supplement (DFARS) Part 204, Administrative Matters, and related clauses...

  17. 76 FR 72916 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department of... Acquisition Regulation Supplement (DFARS) Part 204, Administrative Matters: U.S. International Atomic...

  18. 76 FR 35424 - Information Collection Requirements; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-17

    ... Regulation Supplement; Construction and Architect-Engineer Contracts AGENCY: Defense Acquisition Regulations... Acquisition Regulation Supplement (DFARS) Part 236, Construction and Architect-Engineer Contracts, and...

  19. Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth

    PubMed Central

    Hohm, Tim; Demarsy, Emilie; Quan, Clément; Allenbach Petrolati, Laure; Preuten, Tobias; Vernoux, Teva; Bergmann, Sven; Fankhauser, Christian

    2014-01-01

    Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H+-ATPases that are required to control apoplastic pH. Our results show that H+-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H+-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH. PMID:25261457

  20. Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

    SciTech Connect

    Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

    2006-05-25

    Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

  1. TNL-mediated immunity in Arabidopsis requires complex regulation of the redundant ADR1 gene family.

    PubMed

    Dong, Oliver Xiaoou; Tong, Meixuezi; Bonardi, Vera; El Kasmi, Farid; Woloshen, Virginia; Wünsch, Lisa K; Dangl, Jeffery L; Li, Xin

    2016-05-01

    Nucleotide-binding leucine-rich repeat proteins (NLRs) serve as intracellular immune receptors in animals and plants. Sensor NLRs perceive pathogen-derived effector molecules and trigger robust host defense. Recent studies revealed the role of three coiled-coil-type NLRs (CNLs) of the ADR1 family - ADR1, ADR1-L1 and ADR1-L2 - as redundant helper NLRs, whose function is required for defense mediated by multiple sensor NLRs. From a mutant snc1-enhancing (MUSE) forward genetic screen in Arabidopsis targeted to identify negative regulators of snc1 that encodes a TIR-type NLR (TNL), we isolated two alleles of muse15, both carrying mutations in ADR1-L1. Interestingly, loss of ADR1-L1 also enhances immunity-related phenotypes in other autoimmune mutants including cpr1, bal and lsd1. This immunity-enhancing effect is not mediated by increased SNC1 protein stability, nor is it fully dependent on the accumulation of the defense hormone salicylic acid (SA). Transcriptional analysis revealed an upregulation of ADR1 and ADR1-L2 in the adr1-L1 background, which may overcompensate the loss of ADR1-L1, resulting in enhanced immunity. Interestingly, autoimmunity of snc1 and chs2, which encode typical TNLs, is fully suppressed by the adr1 triple mutant, suggesting that the ADRs are required for TNL downstream signaling. This study extends our knowledge on the interplay among ADRs and reveals their complexity in defense regulation. PMID:27074399

  2. Hic-5 is required for myofibroblast differentiation by regulating mechanically dependent MRTF-A nuclear accumulation.

    PubMed

    Varney, Scott D; Betts, Courtney B; Zheng, Rui; Wu, Lei; Hinz, Boris; Zhou, Jiliang; Van De Water, Livingston

    2016-02-15

    How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-β on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-β. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-β. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-β. PMID:26759173

  3. Proper gibberellin localization in vascular tissue is required to regulate adventitious root development in tobacco.

    PubMed

    Niu, Shihui; Li, Zhexin; Yuan, Huwei; Fang, Pan; Chen, Xiaoyang; Li, Wei

    2013-08-01

    Bioactive gibberellins (GAs) are involved in many developmental aspects of the life cycle of plants, acting either directly or through interaction with other hormones. Accumulating evidence suggests that GAs have an important effect on root growth; however, there is currently little information on the specific regulatory mechanism of GAs during adventitious root development. A study was conducted on tobacco (Nicotiana tabacum) plants for altered rates of biosynthesis, catabolism, and GA signalling constitutively or in specific tissues using a transgenic approach. In the present study, PtGA20ox, PtGA2ox1, and PtGAI were overexpressed under the control of the 35S promoter, vascular cambium-specific promoter (LMX5), or root meristem-specific promoter (TobRB7), respectively. Evidence is provided that the precise localization of bioactive GA in the stem but not in the roots is required to regulate adventitious root development in tobacco. High levels of GA negatively regulate the early initiation step of root formation through interactions with auxin, while a proper and mobile GA signal is required for the emergence and subsequent long-term elongation of established primordia. The results demonstrated that GAs have an inhibitory effect on adventitious root formation but a stimulatory effect on root elongation. PMID:23918971

  4. Endocytosis-dependent coordination of multiple actin regulators is required for wound healing

    PubMed Central

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla

    2015-01-01

    The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form “signaling centers” along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing. PMID:26216900

  5. Acinetobacter baumannii Response to Host-Mediated Zinc Limitation Requires the Transcriptional Regulator Zur

    PubMed Central

    Mortensen, Brittany L.; Rathi, Subodh; Chazin, Walter J.

    2014-01-01

    Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia in intensive care units, and the increasing rates of antibiotic resistance make treating these infections challenging. Consequently, there is an urgent need to develop new antimicrobials to treat A. baumannii infections. One potential therapeutic option is to target bacterial systems involved in maintaining appropriate metal homeostasis, processes that are critical for the growth of pathogens within the host. The A. baumannii inner membrane zinc transporter ZnuABC is required for growth under low-zinc conditions and for A. baumannii pathogenesis. The expression of znuABC is regulated by the transcriptional repressor Zur. To investigate the role of Zur during the A. baumannii response to zinc limitation, a zur deletion mutant was generated, and transcriptional changes were analyzed using RNA sequencing. A number of Zur-regulated genes were identified that exhibit increased expression both when zur is absent and under low-zinc conditions, and Zur binds to predicted Zur box sequences of several genes affected by zinc levels or the zur mutation. Furthermore, the zur mutant is impaired for growth in the presence of both high and low zinc levels compared to wild-type A. baumannii. Finally, the zur mutant exhibits a defect in dissemination in a mouse model of A. baumannii pneumonia, establishing zinc sensing as a critical process during A. baumannii infection. These results define Zur-regulated genes within A. baumannii and demonstrate a requirement for Zur in the A. baumannii response to the various zinc levels experienced within the vertebrate host. PMID:24816603

  6. UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development

    PubMed Central

    Kent, Brandon; Magnani, Elena; Walsh, Martin J.; Sadler, Kirsten C.

    2016-01-01

    Landmark epigenetic events underlie early embryonic development, yet how epigenetic modifiers are regulated to achieve rapid epigenome re-patterning is not known. Uhrf1 and DNA methyltransferase 1 (Dnmt1) are known to largely mediate maintenance DNA methylation and Uhrf1 is also required for both Dnmt1 localization and stability. Here, we investigate how these two key epigenetic modifiers regulate early zebrafish development and characterize the developmental consequences of disrupting their homeostatic relationship. Unlike Uhrf1 knockdown, which causes developmental arrest and death prior to gastrulation, overexpression of human UHRF1 (WT-UHRF1) caused asymmetric epiboly, inefficient gastrulation and multi-systemic defects. UHRF1 phosphorylation was previously demonstrated as essential for zebrafish embryogenesis, and we found that penetrance of the asymmetric epiboly phenotype was significantly increased in embryos injected with mRNA encoding non-phosphorylatable UHRF1 (UHRF1S661A). Surprisingly, both WT-UHRF1 and UHRF1S661A overexpression caused DNA hypomethylation. However, since other approaches that caused an equivalent degree of DNA hypomethylation did not cause the asymmetric epiboly phenotype, we conclude that bulk DNA methylation is not the primary mechanism. Instead, UHRF1S661A overexpression resulted in accumulation of Dnmt1 protein and the overexpression of both WT and a catalytically inactive Dnmt1 phenocopied the WT-UHRF1 overexpressing embryos. Dnmt1 knockdown suppressed the phenotype caused by UHRF1S661A overexpression, and Uhrf1 knockdown suppressed the effect of Dnmt1 overexpression. Therefore, we conclude that the interaction between these two proteins is the mechanism underlying the observed developmental defects. This indicates that Dnmt1 stability requires UHRF1 phosphorylation and that crosstalk between the proteins is essential for the function of these two important epigenetic regulators during gastrulation. PMID:26851214

  7. Telomere Length Regulation and Telomeric Chromatin Require the Nonsense-Mediated mRNA Decay Pathway

    PubMed Central

    Lew, Jodi E.; Enomoto, Shinichiro; Berman, Judith

    1998-01-01

    Rap1p localization factor 4 (RLF4) is a Saccharomyces cerevisiae gene that was identified in a screen for mutants that affect telomere function and alter the localization of the telomere binding protein Rap1p. In rlf4 mutants, telomeric silencing is reduced and telomere DNA tracts are shorter, indicating that RLF4 is required for both the establishment and/or maintenance of telomeric chromatin and for the control of telomere length. In this paper, we demonstrate that RLF4 is allelic to NMD2/UPF2, a gene required for the nonsense-mediated mRNA decay (NMD) pathway (Y. Cui, K. W. Hagan, S. Zhang, and S. W. Peltz, Mol. Cell. Biol. 9:423–436, 1995, and F. He and A. Jacobson, Genes Dev. 9:437–454, 1995). The NMD pathway, which requires Nmd2p/Rlf4p together with two other proteins, (Upf1p and Upf3p), targets nonsense messages for degradation in the cytoplasm by the exoribonuclease Xrn1p. Deletion of UPF1 and UPF3 caused telomere-associated defects like those caused by rlf4 mutations, implying that the NMD pathway, rather than an NMD-independent function of Nmd2p/Rlf4p, is required for telomere functions. In addition, telomere length regulation required Xrn1p but not Rat1p, a nuclear exoribonuclease with functional similarity to Xrn1p (A. W. Johnson, Mol. Cell. Biol. 17:6122–6130, 1997). In contrast, telomere-associated defects were not observed in pan2, pan3, or pan2 pan3 strains, which are defective in the intrinsic deadenylation-dependent decay of normal (as opposed to nonsense) mRNAs. Thus, loss of the NMD pathway specifically causes defects at telomeres, demonstrating a physiological requirement for the NMD pathway in normal cell functions. We propose a model in which the NMD pathway regulates the levels of specific mRNAs that are important for telomere functions. PMID:9742129

  8. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  9. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  10. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture Regulations... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

  11. 43 CFR 1821.12 - Are these the only regulations that will apply to my application or other required document?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false Are these the only regulations that will apply to my application or other required document? 1821.12 Section 1821.12 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR GENERAL MANAGEMENT (1000) APPLICATION...

  12. 43 CFR 1821.12 - Are these the only regulations that will apply to my application or other required document?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 2 2012-10-01 2012-10-01 false Are these the only regulations that will apply to my application or other required document? 1821.12 Section 1821.12 Public Lands: Interior Regulations Relating to Public Lands (Continued) BUREAU OF LAND MANAGEMENT, DEPARTMENT OF THE INTERIOR...

  13. Transcriptional Regulation via Nuclear Receptor Crosstalk Required for the Drosophila Circadian Clock

    PubMed Central

    Jaumouillé, Edouard; Machado Almeida, Pedro; Stähli, Patrick; Koch, Rafael; Nagoshi, Emi

    2015-01-01

    Summary Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and CLOCK/CYCLE (CLK/CYC) (in Drosophila) drive the expression of the period (per) family genes. The PER-containing complexes inhibit the activity of CLOCK/BMAL1 or CLK/CYC, thereby forming a negative feedback loop [1]. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [2]. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. We show here that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor UNF are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and UNF bind to per regulatory sequences and act together to enhance the CLK/CYC-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. Our results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks. PMID:26004759

  14. DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts

    PubMed Central

    Mendieta, Jesús; Pérez-Lago, Laura; Salas, Margarita; Camacho, Ana

    2007-01-01

    The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4–DNA complex contains two identical protomers that make similar interactions with the target sequence 5′-AACTTTTT-15 bp-AAAATGTT-3′. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated binding sites. The binding specificity depends critically on base pairs of the target sequence through both direct as well as indirect readout. There is only one specific contact between a base and an amino acid residue; other contacts take place with the phosphate backbone. Alteration of direct amino acid–base contacts, or mutation of non-contacted A·T base pairs at A-tracts abolished binding. We generated three 5 ns molecular dynamics (MD) simulations to investigate the basis for the p4–DNA complex specificity. Recognition is controlled by the protein and depends on DNA dynamic properties. MD results on protein–DNA contacts and the divergence of p4 affinity to modified binding sites reveal an inherent asymmetry, which is required for p4-specific binding and may be crucial for transcription regulation. PMID:17452358

  15. Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock.

    PubMed

    Jaumouillé, Edouard; Machado Almeida, Pedro; Stähli, Patrick; Koch, Rafael; Nagoshi, Emi

    2015-06-01

    Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and CLOCK/CYCLE (CLK/CYC) (in Drosophila) drive the expression of the period (per) family genes. The PER-containing complexes inhibit the activity of CLOCK/BMAL1 or CLK/CYC, thereby forming a negative feedback loop [1]. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [2]. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. We show here that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor UNF are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and UNF bind to per regulatory sequences and act together to enhance the CLK/CYC-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. Our results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks. PMID:26004759

  16. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  17. Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis

    PubMed Central

    Aitchison, Adam J.; Arsenault, Daniel J.; Ridgway, Neale D.

    2015-01-01

    The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs. PMID:26108622

  18. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

    PubMed Central

    Christianson, John P.; Flyer-Adams, Johanna G.; Drugan, Robert C.; Amat, Jose; Daut, Rachel A.; Foilb, Allison R.; Watkins, Linda R.; Maier, Steven F.

    2014-01-01

    Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed “behavioral immunization”. Recent data implicate protein synthesis within the ventromedial prefrontal cortex (mPFC) as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and 1 week later to uncontrollable tailshocks, followed 24 h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDARs) and the extracellular signal-regulated kinase (ERK) cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL) or infralimbic (IL) mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent signaling within the PL region is required for behavioral immunization, a learned form of stressor resistance. PMID:25324750

  19. 78 FR 70294 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-25

    ... Regulation Supplement; Administrative Matters AGENCY: Defense Acquisition Regulations System, Department of..., Administrative Matters, and related clauses at DFARS 252.204; DD Form 2051, Request for Assignment of...

  20. 75 FR 47561 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-06

    ... Regulation Supplement; Contract Financing (OMB Control Number 0704- 0359) AGENCY: Defense Acquisition... and OMB Number: Defense Federal Acquisition Regulation Supplement (DFARS) part 232, Contract...

  1. Sequence Requirements for Myosin Gene Expression and Regulation in Caenorhabditis Elegans

    PubMed Central

    Okkema, P. G.; Harrison, S. W.; Plunger, V.; Aryana, A.; Fire, A.

    1993-01-01

    Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers. PMID:8244003

  2. Nutritional requirements and nitrogen-dependent regulation of proteinase activity of Lactobacillus helveticus CRL 1062.

    PubMed

    Hebert, E M; Raya, R R; De Giori, G S

    2000-12-01

    The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or beta-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%. PMID:11097908

  3. The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function

    PubMed Central

    Gehman, Lauren T.; Meera, Pratap; Stoilov, Peter; Shiue, Lily; O'Brien, Janelle E.; Meisler, Miriam H.; Ares, Manuel; Otis, Thomas S.; Black, Douglas L.

    2012-01-01

    The Rbfox proteins (Rbfox1, Rbfox2, and Rbfox3) regulate the alternative splicing of many important neuronal transcripts and have been implicated in a variety of neurological disorders. However, their roles in brain development and function are not well understood, in part due to redundancy in their activities. Here we show that, unlike Rbfox1 deletion, the CNS-specific deletion of Rbfox2 disrupts cerebellar development. Genome-wide analysis of Rbfox2−/− brain RNA identifies numerous splicing changes altering proteins important both for brain development and mature neuronal function. To separate developmental defects from alterations in the physiology of mature cells, Rbfox1 and Rbfox2 were deleted from mature Purkinje cells, resulting in highly irregular firing. Notably, the Scn8a mRNA encoding the Nav1.6 sodium channel, a key mediator of Purkinje cell pacemaking, is improperly spliced in RbFox2 and Rbfox1 mutant brains, leading to highly reduced protein expression. Thus, Rbfox2 protein controls a post-transcriptional program required for proper brain development. Rbfox2 is subsequently required with Rbfox1 to maintain mature neuronal physiology, specifically Purkinje cell pacemaking, through their shared control of sodium channel transcript splicing. PMID:22357600

  4. The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function.

    PubMed

    Gehman, Lauren T; Meera, Pratap; Stoilov, Peter; Shiue, Lily; O'Brien, Janelle E; Meisler, Miriam H; Ares, Manuel; Otis, Thomas S; Black, Douglas L

    2012-03-01

    The Rbfox proteins (Rbfox1, Rbfox2, and Rbfox3) regulate the alternative splicing of many important neuronal transcripts and have been implicated in a variety of neurological disorders. However, their roles in brain development and function are not well understood, in part due to redundancy in their activities. Here we show that, unlike Rbfox1 deletion, the CNS-specific deletion of Rbfox2 disrupts cerebellar development. Genome-wide analysis of Rbfox2(-/-) brain RNA identifies numerous splicing changes altering proteins important both for brain development and mature neuronal function. To separate developmental defects from alterations in the physiology of mature cells, Rbfox1 and Rbfox2 were deleted from mature Purkinje cells, resulting in highly irregular firing. Notably, the Scn8a mRNA encoding the Na(v)1.6 sodium channel, a key mediator of Purkinje cell pacemaking, is improperly spliced in RbFox2 and Rbfox1 mutant brains, leading to highly reduced protein expression. Thus, Rbfox2 protein controls a post-transcriptional program required for proper brain development. Rbfox2 is subsequently required with Rbfox1 to maintain mature neuronal physiology, specifically Purkinje cell pacemaking, through their shared control of sodium channel transcript splicing. PMID:22357600

  5. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    PubMed

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  6. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling

    PubMed Central

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  7. Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure.

    PubMed

    Gatto, Cheryl L; Broadie, Kendal

    2008-08-01

    Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function. PMID:18579676

  8. Temporal Requirements of the Fragile X Mental Retardation Protein in the Regulation of Synaptic Structure

    PubMed Central

    Gatto, Cheryl L.; Broadie, Kendal

    2009-01-01

    Fragile X Syndrome (FraX), caused by the loss of function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is critical to define when and where FMRP loss causes the manifestation of synaptic defects, and whether reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We have used the Gene-Switch (GS) method to conditionally drive dFMRP to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in dfmr1 null mutants, demonstrating a presynaptic requirement for synapse structuring. In contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1 null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function. PMID:18579676

  9. Profiles of Motivated Self-Regulation in College Computer Science Courses: Differences in Major versus Required Non-Major Courses

    ERIC Educational Resources Information Center

    Shell, Duane F.; Soh, Leen-Kiat

    2013-01-01

    The goal of the present study was to utilize a profiling approach to understand differences in motivation and strategic self-regulation among post-secondary STEM students in major versus required non-major computer science courses. Participants were 233 students from required introductory computer science courses (194 men; 35 women; 4 unknown) at…

  10. 76 FR 36905 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Taxes

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-23

    ... Regulation Supplement; Taxes AGENCY: Defense Acquisition Regulations System, Department of Defense (DoD... Supplement (DFARS) Part 229, Taxes, and related clause at DFARS 252.229-7010; OMB Control Number...

  11. 76 FR 67152 - Federal Acquisition Regulation; Submission for OMB Review; Cost or Pricing Data Requirements and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-31

    ... in the Federal Register at 76 FR 35218, on June 16, 2011. No comments were received. Public comments... Pricing Data, by any of the following methods: Regulations.gov : http://www.regulations.gov . Sub...

  12. Requirement for non-regulated, constitutive calcium influx in macrophage survival signaling

    SciTech Connect

    Tano, Jean-Yves; Vazquez, Guillermo

    2011-04-08

    Highlights: {yields} We examine the role of constitutive Ca{sup 2+} influx in macrophage survival. {yields} Survival signaling exhibits a mandatory requirement for constitutive Ca{sup 2+} influx. {yields} CAM/CAMKII couples constitutive Ca{sup 2+} influx to survival signaling. -- Abstract: The phosphatidylinositol-3-kinase (PI3K)/AKT axis and the Nuclear Factor kappa B (NF{kappa}B) pathway play critical roles in macrophage survival. In cells other than macrophages proper operation of those two pathways requires Ca{sup 2+} influx into the cell, but if that is the case in macrophages remains unexplored. In the present work we used THP-1-derived macrophages and a pharmacological approach to examine for the first time the role of constitutive, non-regulated Ca{sup 2+} influx in PI3K/AKT and NF{kappa}B signaling. Blocking constitutive function of Ca{sup 2+}-permeable channels with the organic channel blocker SKF96365 completely prevented phosphorylation of I{kappa}B{alpha}, AKT and its downstream target BAD in TNF{alpha}-treated macrophages. A similar effect was observed upon treating macrophages with the calmodulin (CAM) inhibitor W-7 or the calmodulin-dependent kinase II (CAMKII) inhibitor KN-62. In addition, pre-treating macrophages with SKF96365 significantly enhanced TNF{alpha}-induced apoptosis. Our findings suggest that in THP-1-derived macrophages survival signaling depends, to a significant extent, on constitutive Ca{sup 2+} influx presumably through a mechanism that involves the CAM/CAMKII axis as a coupling component between constitutive Ca{sup 2+} influx and activation of survival signaling.

  13. 77 FR 58817 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-24

    ... Regulation Supplement (DFARS); Contract Financing AGENCY: Defense Acquisition Regulations System, Department... comments. Email: dfars@osd.mil . Include OMB Control Number 0704-0321 in the subject line of the message..., Associated Form, and OMB Number: Defense Federal Acquisition Regulation Supplement (DFARS) Part 232,...

  14. 78 FR 68829 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-15

    ... Regulation Supplement; Contract Financing AGENCY: Defense Acquisition Regulations System, Department of.... Email: dfars@osd.mil . Include OMB Control Number 0704-0359 in the subject line of the message. Fax... Federal Acquisition Regulation Supplement (DFARS) Part 232, Contract Financing, and related clause...

  15. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

    PubMed

    Zhang, Jixiang; Xie, Shaojun; Cheng, Jinkui; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2016-06-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  16. Multiple protein kinase A-regulated events are required for transcriptional induction by cAMP.

    PubMed Central

    Brindle, P; Nakajima, T; Montminy, M

    1995-01-01

    The second messenger cAMP stimulates the expression of numerous genes via the protein kinase A-mediated phosphorylation of the cAMP response element-binding protein (CREB) at Ser-133. Ser-133 phosphorylation, in turn, appears to induce target gene expression by promoting interaction between CREB and CBP, a 265-kDa nuclear phospho-CREB-binding protein. It is unclear, however, whether Ser-133 phosphorylation per se is sufficient for CREB-CBP complex formation and for target gene induction in vivo. Here we examine CREB activity in Jurkat T cells after stimulation of the T-cell receptor (TCR), an event that leads to calcium entry and diacylglycerol production. Triggering of the TCR stimulated Ser-133 phosphorylation of CREB with high stoichiometry, but TCR activation did not promote CREB-CBP complex formation or target gene induction unless suboptimal doses of cAMP agonist were provided as a costimulus. Our results demonstrate that, in addition to mediating Ser-133 phosphorylation of CREB, protein kinase A regulates additional proteins that are required for recruitment of the transcriptional apparatus to cAMP-responsive genes. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479832

  17. Brg1 directly regulates Olig2 transcription and is required for oligodendrocyte progenitor cell specification.

    PubMed

    Matsumoto, Steven; Banine, Fatima; Feistel, Kerstin; Foster, Scott; Xing, Rubing; Struve, Jaime; Sherman, Larry S

    2016-05-15

    The Olig2 basic-helix-loop-helix transcription factor promotes oligodendrocyte specification in early neural progenitor cells (NPCs), including radial glial cells, in part by recruiting SWI/SNF chromatin remodeling complexes to the enhancers of genes involved in oligodendrocyte differentiation. How Olig2 expression is regulated during oligodendrogliogenesis is not clear. Here, we find that the Brg1 subunit of SWI/SNF complexes interacts with a proximal Olig2 promoter and represses Olig2 transcription in the mouse cortex at E14, when oligodendrocyte progenitors (OPCs) are not yet found in this location. Brg1 does not interact with the Olig2 promoter in the E14 ganglionic eminence, where NPCs differentiate into Olig2-positive OPCs. Consistent with these findings, Brg1-null NPCs demonstrate precocious expression of Olig2 in the cortex. However, these cells fail to differentiate into OPCs. We further find that Brg1 is necessary for neuroepithelial-to-radial glial cell transition, but not neuronal differentiation despite a reduction in expression of the pro-neural transcription factor Pax6. Collectively, these and earlier findings support a model whereby Brg1 promotes neurogenic radial glial progenitor cell specification but is dispensable for neuronal differentiation. Concurrently, Brg1 represses Olig2 expression and the specification of OPCs, but is required for OPC differentiation and oligodendrocyte maturation. PMID:27067865

  18. MT1-MMP is required for myeloid cell fusion via regulation of Rac1 signaling

    PubMed Central

    Gonzalo, Pilar; Guadamillas, Marta C.; Hernández-Riquer, Mª Victoria; Pollán, Ángela; Grande-García, Araceli; Bartolomé, Rubén A.; Vasanji, Amit; Ambrogio, Chiara; Chiarle, Roberto; Teixidó, Joaquín; Risteli, Juha; Apte, Suneel S.; del Pozo, Miguel A.; Arroyo, Alicia G.

    2009-01-01

    SUMMARY Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse in various circumstances but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP-null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas. PMID:20152179

  19. Retinophilin is a light-regulated phosphoprotein required to suppress photoreceptor dark noise in Drosophila

    PubMed Central

    Mecklenburg, Kirk L.; Takemori, Nobuaki; Komori, Naoka; Chu, Brian; Hardie, Roger C.; Matsumoto, Hiroyuki; O’Tousa, Joseph. E.

    2010-01-01

    Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP/CG10233) in Drosophila photoreceptors, and establish its involvement in dark noise suppression. RTP possesses MORN (Membrane Occupation and Recognition Nexus) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking NINAC myosin III, a motor protein/kinase, also display a similar dark noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is due to lack of RTP, and further, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors. PMID:20107052

  20. Novel insights into iron regulation and requirement in marine medaka Oryzias melastigma.

    PubMed

    Wang, Jian; Wang, Wen-Xiong

    2016-01-01

    Iron (Fe) is an essential trace element for marine fish. However, our knowledge of Fe requirements at different development stages of marine fish is still limited. Here, we reported the efficient Fe absorption strategies adopted by larval fish under different dietary Fe supplementary levels (i.e., 0-640 mg/kg). Biokinetically, the larval fish controlled their dietary Fe assimilation efficiency (AE, 1.6-18.5%), and enhanced their waterborne Fe uptake (ca. 2.5 fold change of uptake rate constant) once the dietary Fe was deficient (i.e., 27.4 mg Fe/kg feed). Transcriptionally, the expression of hepcidin1 (hep1; Fe regulator; i.e., 2.3-15.7 fold change) in larval fish was positively correlated with the Fe supplementary levels. Comparatively, the female adult fish were poor in assimilating the added Fe source (i.e., ferric form) with similar life-sustainable levels of Fe (i.e., 0.046-0.12 μg/g/d assimilated for Fe supplementary levels of 27.4, 162 and 657 mg Fe/kg feed). The overall feeding experiments suggested that dietary net Fe flux sufficient for the normal growth of larval medaka was 0.71-1.75 μg/g/d (i.e., 83.9 mg Fe/kg feed), consistent with the modeled value (i.e., 1.09-2.16 μg/g/d). In female adults, the estimated essential net Fe flux was 0.88-0.90 μg/g/d. PMID:27216705

  1. Novel insights into iron regulation and requirement in marine medaka Oryzias melastigma

    NASA Astrophysics Data System (ADS)

    Wang, Jian; Wang, Wen-Xiong

    2016-05-01

    Iron (Fe) is an essential trace element for marine fish. However, our knowledge of Fe requirements at different development stages of marine fish is still limited. Here, we reported the efficient Fe absorption strategies adopted by larval fish under different dietary Fe supplementary levels (i.e., 0–640 mg/kg). Biokinetically, the larval fish controlled their dietary Fe assimilation efficiency (AE, 1.6–18.5%), and enhanced their waterborne Fe uptake (ca. 2.5 fold change of uptake rate constant) once the dietary Fe was deficient (i.e., 27.4 mg Fe/kg feed). Transcriptionally, the expression of hepcidin1 (hep1; Fe regulator; i.e., 2.3–15.7 fold change) in larval fish was positively correlated with the Fe supplementary levels. Comparatively, the female adult fish were poor in assimilating the added Fe source (i.e., ferric form) with similar life-sustainable levels of Fe (i.e., 0.046–0.12 μg/g/d assimilated for Fe supplementary levels of 27.4, 162 and 657 mg Fe/kg feed). The overall feeding experiments suggested that dietary net Fe flux sufficient for the normal growth of larval medaka was 0.71–1.75 μg/g/d (i.e., 83.9 mg Fe/kg feed), consistent with the modeled value (i.e., 1.09–2.16 μg/g/d). In female adults, the estimated essential net Fe flux was 0.88–0.90 μg/g/d.

  2. Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae.

    PubMed Central

    Neiman, A M; Katz, L; Brennwald, P J

    2000-01-01

    Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells. PMID:10924463

  3. NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation

    SciTech Connect

    Adachi, Atsuo; Takahashi, Tomosaburo; Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko; Ueyama, Tomomi; Matsubara, Hiroaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

  4. Novel insights into iron regulation and requirement in marine medaka Oryzias melastigma

    PubMed Central

    Wang, Jian; Wang, Wen-Xiong

    2016-01-01

    Iron (Fe) is an essential trace element for marine fish. However, our knowledge of Fe requirements at different development stages of marine fish is still limited. Here, we reported the efficient Fe absorption strategies adopted by larval fish under different dietary Fe supplementary levels (i.e., 0–640 mg/kg). Biokinetically, the larval fish controlled their dietary Fe assimilation efficiency (AE, 1.6–18.5%), and enhanced their waterborne Fe uptake (ca. 2.5 fold change of uptake rate constant) once the dietary Fe was deficient (i.e., 27.4 mg Fe/kg feed). Transcriptionally, the expression of hepcidin1 (hep1; Fe regulator; i.e., 2.3–15.7 fold change) in larval fish was positively correlated with the Fe supplementary levels. Comparatively, the female adult fish were poor in assimilating the added Fe source (i.e., ferric form) with similar life-sustainable levels of Fe (i.e., 0.046–0.12 μg/g/d assimilated for Fe supplementary levels of 27.4, 162 and 657 mg Fe/kg feed). The overall feeding experiments suggested that dietary net Fe flux sufficient for the normal growth of larval medaka was 0.71–1.75 μg/g/d (i.e., 83.9 mg Fe/kg feed), consistent with the modeled value (i.e., 1.09–2.16 μg/g/d). In female adults, the estimated essential net Fe flux was 0.88–0.90 μg/g/d. PMID:27216705

  5. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 2 2012-04-01 2012-04-01 false If a tribe holds a certificate of self-regulation, is it... REGULATION OF CLASS II GAMING § 518.7 If a tribe holds a certificate of self-regulation, is it required to... certificate of self-regulation shall be required to submit a self-regulation report annually to the...

  6. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 2 2013-04-01 2013-04-01 false If a tribe holds a certificate of self-regulation, is it... REGULATION OF CLASS II GAMING § 518.7 If a tribe holds a certificate of self-regulation, is it required to... certificate of self-regulation shall be required to submit a self-regulation report annually to the...

  7. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 2 2011-04-01 2011-04-01 false If a tribe holds a certificate of self-regulation, is it... REGULATION OF CLASS II GAMING § 518.7 If a tribe holds a certificate of self-regulation, is it required to... certificate of self-regulation shall be required to submit a self-regulation report annually to the...

  8. 75 FR 26165 - Regulation of Fuels and Fuel Additives: Alternative Affirmative Defense Requirements for Ultra...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-11

    ...EPA is issuing a proposed rule to amend the diesel sulfur regulations to allow refiners, importers, distributors, and retailers of highway diesel fuel the option to use an alternative affirmative defense if the Agency finds highway diesel fuel samples above the specified sulfur standard at retail facilities. This rule also proposes to amend the gasoline benzene regulations to allow......

  9. 78 FR 30898 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Rights in...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-23

    ... Regulation Supplement; Rights in Technical Data and Computer Software AGENCY: Defense Acquisition Regulations... in Technical Data, and Subpart 227.72, Rights in Computer Software and Computer Software... are associated with rights in technical data and computer software. DoD needs this information...

  10. 78 FR 67132 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Contract...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-08

    ... Regulation Supplement; Contract Pricing AGENCY: Defense Acquisition Regulations System, Department of Defense... comments. Email: dfars@osd.mil . Include OMB Control Number 0704-0232 in the subject line of the message...) Subpart 215.4, Contract Pricing; DD Form 1861, Contract Facilities Capital Cost of Money; OMB...

  11. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of... 7 Agriculture 5 2014-01-01 2014-01-01 false Movement of plant pests regulated; permits...

  12. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of... 7 Agriculture 5 2013-01-01 2013-01-01 false Movement of plant pests regulated; permits...

  13. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of... 7 Agriculture 5 2011-01-01 2011-01-01 false Movement of plant pests regulated; permits...

  14. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of... 7 Agriculture 5 2012-01-01 2012-01-01 false Movement of plant pests regulated; permits...

  15. 7 CFR 330.200 - Movement of plant pests regulated; permits required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE FEDERAL PLANT PEST REGULATIONS; GENERAL; PLANT PESTS; SOIL, STONE, AND QUARRY PRODUCTS; GARBAGE Movement of Plant Pests § 330.200 Movement of... 7 Agriculture 5 2010-01-01 2010-01-01 false Movement of plant pests regulated; permits...

  16. Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes*

    PubMed Central

    Tate, Jennifer J.; Cooper, Terrance G.

    2013-01-01

    Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. PMID:23935103

  17. 77 FR 33638 - Amendment to the Bank Secrecy Act Regulations-Exemption From the Requirement To Report...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-07

    ...FinCEN is issuing this final rule to amend the regulations that allow depository institutions to exempt transactions of certain payroll customers \\1\\ from the requirement to report transactions in currency in excess of $10,000. The rule substitutes the term ``frequently'' for ``regularly'' in the provision of the exemption rules dealing with payroll customers. This modification of the......

  18. 77 FR 72970 - Revisions to Stormwater Regulations To Clarify That an NPDES Permit Is Not Required for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-07

    ...The EPA is revising its Phase I stormwater regulations to clarify that stormwater discharges from logging roads do not constitute stormwater discharges associated with industrial activity and that a National Pollutant Discharge Elimination System (NPDES) permit is not required for these stormwater...

  19. Immigration Rules, Regulations, and Requirements and Foreign Student Enrollments. Southeast Florida Education Consortium Workshop (Miami, Florida, February 18, 1981).

    ERIC Educational Resources Information Center

    Matthewson, Douglas E., Jr., Comp.

    Information is presented about a workshop on immigration documents, rules, regulations, and requirements associated with international students and nonimmigrant alien students. Twenty-six members of the Southeast Florida Educational Consortium participated, representing campus admissions and registrars offices, international student programs, and…

  20. 7 CFR 301.76-4 - Labeling requirements for regulated nursery stock produced within an area quarantined for citrus...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... OF AGRICULTURE DOMESTIC QUARANTINE NOTICES Citrus Greening and Asian Citrus Psyllid § 301.76-4 Labeling requirements for regulated nursery stock produced within an area quarantined for citrus greening... produced within an area quarantined for citrus greening. 301.76-4 Section 301.76-4 Agriculture...

  1. 75 FR 35338 - Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-22

    ...-AB07 Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of... participants regarding compliance. In enacting Title XI of the Food, Conservation and Energy Act of 2008 (Farm..., and its coverage was to encompass the complete chain of commerce and give the Secretary of...

  2. Caveolin-1–dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo

    PubMed Central

    Marchiando, Amanda M.; Shen, Le; Graham, W. Vallen; Weber, Christopher R.; Schwarz, Brad T.; Austin, Jotham R.; Raleigh, David R.; Guan, Yanfang; Watson, Alastair J.M.; Montrose, Marshall H.

    2010-01-01

    Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor (α) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1–dependent endocytosis of occludin to effect structural and functional tight junction regulation. PMID:20351069

  3. 49 CFR 379.11 - Waiver of requirements of the regulations in this part.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... from any provision of the regulations in this part may be made by the Secretary upon his/her own initiative or upon submission of a written request by the company. Each request for waiver shall demonstrate... company....

  4. 49 CFR 379.11 - Waiver of requirements of the regulations in this part.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... from any provision of the regulations in this part may be made by the Secretary upon his/her own initiative or upon submission of a written request by the company. Each request for waiver shall demonstrate... company....

  5. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  6. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  7. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  8. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  9. 10 CFR 150.31 - Requirements for Agreement State regulation of byproduct material.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... with the requirements in appendix A of 10 CFR part 40 of this chapter to the maximum extent practicable... material, an Agreement State shall require: (1) Compliance with requirements in appendix A of 10 CFR part... in appendix A of 10 CFR part 40 of this chapter adopted and enforced by the Commission for the...

  10. Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence

    PubMed Central

    Leistikow, Rachel L.; Ramakrishnan, Pavithra; Voskuil, Martin I.; McKinney, John D.

    2016-01-01

    The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ΔpstA1ΔpstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ΔpstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ΔpstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions. PMID:27557082

  11. Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 Is Not Required for Gene Regulation or Virulence.

    PubMed

    Tischler, Anna D; Leistikow, Rachel L; Ramakrishnan, Pavithra; Voskuil, Martin I; McKinney, John D

    2016-01-01

    The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ΔpstA1ΔpstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ΔpstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ΔpstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions. PMID:27557082

  12. Transcription factors of Lotus: regulation of isoflavonoid biosynthesis requires coordinated changes in transcription factor activity.

    PubMed

    Shelton, Dale; Stranne, Maria; Mikkelsen, Lisbeth; Pakseresht, Nima; Welham, Tracey; Hiraka, Hideki; Tabata, Satoshi; Sato, Shusei; Paquette, Suzanne; Wang, Trevor L; Martin, Cathie; Bailey, Paul

    2012-06-01

    Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors. PMID:22529285

  13. Profiles of Motivated Self-Regulation in College Computer Science Courses: Differences in Major versus Required Non-Major Courses

    NASA Astrophysics Data System (ADS)

    Shell, Duane F.; Soh, Leen-Kiat

    2013-12-01

    The goal of the present study was to utilize a profiling approach to understand differences in motivation and strategic self-regulation among post-secondary STEM students in major versus required non-major computer science courses. Participants were 233 students from required introductory computer science courses (194 men; 35 women; 4 unknown) at a large Midwestern state university. Cluster analysis identified five profiles: (1) a strategic profile of a highly motivated by-any-means good strategy user; (2) a knowledge-building profile of an intrinsically motivated autonomous, mastery-oriented student; (3) a surface learning profile of a utility motivated minimally engaged student; (4) an apathetic profile of an amotivational disengaged student; and (5) a learned helpless profile of a motivated but unable to effectively self-regulate student. Among CS majors and students in courses in their major field, the strategic and knowledge-building profiles were the most prevalent. Among non-CS majors and students in required non-major courses, the learned helpless, surface learning, and apathetic profiles were the most prevalent. Students in the strategic and knowledge-building profiles had significantly higher retention of computational thinking knowledge than students in other profiles. Students in the apathetic and surface learning profiles saw little instrumentality of the course for their future academic and career objectives. Findings show that students in STEM fields taking required computer science courses exhibit the same constellation of motivated strategic self-regulation profiles found in other post-secondary and K-12 settings.

  14. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes.

    PubMed

    Brown, Daren W; Busman, Mark; Proctor, Robert H

    2014-08-01

    The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1, that regulates markedly different genes in different fungi. Despite these differences, many of the regulated genes encode effector proteins or proteins involved in the synthesis of secondary metabolites (SM), both of which can contribute to pathogenicity. Fusarium verticillioides is both an endophyte and a pathogen of maize and can grow as a saprophyte on dead plant material. During growth on live maize plants, the fungus can synthesize a number of toxic SM, including fumonisins, fusarins, and fusaric acid, that can contaminate kernels and kernel-based food and feed. In this study, the role of F. verticillioides SGE1 in pathogenicity and secondary metabolism was examined by gene deletion analysis and transcriptomics. SGE1 is not required for vegetative growth or conidiation but is required for wild-type pathogenicity and affects synthesis of multiple SM, including fumonisins and fusarins. Induced expression of SGE1 enhanced or reduced expression of hundreds of genes, including numerous putative effector genes that could contribute to growth in planta; genes encoding cell surface proteins; gene clusters required for synthesis of fusarins, bikaverin, and an unknown metabolite; as well as the gene encoding the fumonisin cluster transcriptional activator. Together, our results indicate that SGE1 has a role in global regulation of transcription in F. verticillioides that impacts but is not absolutely required for secondary metabolism and pathogenicity on maize. PMID:24742071

  15. 78 FR 63461 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Foreign...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-24

    ... Payments Program Certificate, as prescribed in 225.1101(1), requires an offeror to identify, in its... in 225.1101(4), requires the contractor to provide information on shipping documents and customs... of origin. DFARS 252.225-7020, Trade Agreements Certificate, as prescribed in 225.1101(5),...

  16. 77 FR 12927 - Federal Acquisition Regulation: Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-02

    .... Background DoD, GSA, and NASA published an interim rule in the Federal Register at 76 FR 14548 on March 16... Register at 76 FR 14548 on March 16, 2011, is adopted as final with the following changes: PART 8--REQUIRED...; (5) Competition requirements for establishing BPAs and allowing flexibility in establishing...

  17. 76 FR 17138 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-28

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  18. 76 FR 78933 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-20

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... workshop. The public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  19. 75 FR 51824 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-23

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... workshop. The public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  20. 76 FR 51040 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-17

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... workshop. The public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  1. 77 FR 8886 - Food and Drug Administration Clinical Trial Requirements, Regulations, Compliance, and Good...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-15

    ... HUMAN SERVICES Food and Drug Administration Food and Drug Administration Clinical Trial Requirements... public workshop on FDA's clinical trial requirements is designed to aid the clinical research... interaction with FDA representatives. The program will focus on the relationships among FDA and clinical...

  2. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  3. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  4. 33 CFR 165.921 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in this section— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR... explosives as defined in 49 CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or, for which a permit is required as a condition of a Research and...

  5. A Comparison of the Mandatory Continuing Education (MCE) Requirements of the Regulated Health Occupations in Minnesota.

    ERIC Educational Resources Information Center

    Green-Eide, Beth

    A study reviewed and compared initial and renewal practices for licensure/registration of 13 health care occupations regulated in the state of Minnesota. It examined mandatory continuing education (MCE) documentation and the practices of licensing boards in their enforcement of the MCE legislation. The Minnesota Statutes and Rules for the…

  6. 78 FR 4032 - Prompt Corrective Action, Requirements for Insurance, and Promulgation of NCUA Rules and Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-18

    ... amendment to 12 CFR 791.8. \\1\\ IRPS 03-2, 68 FR 31949 (May 29, 2003). What changes were proposed? On... rule governing the promulgation of regulations.\\9\\ \\6\\ 46 FR 29248 (June 1, 1981). \\7\\ 52 FR 35231 (Sept. 8, 1987). \\8\\ 68 FR at 31949. \\9\\ 12 CFR 791.8(a). When the Board updated its RFA threshold...

  7. 78 FR 20624 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement (DFARS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-05

    ... Regulation Supplement (DFARS); Radio Frequency Identification Advance Shipment Notices AGENCY: Defense... Supplement (DFARS), Part 211 and related clause at 252.211; Radio Frequency Identification Advance Shipment.... Annual Burden Hours: 31,556. Frequency: On occasion. Summary of Information Collection The clause...

  8. 77 FR 51686 - Specialty Crops; Import Regulations; New Pistachio Import Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... in the marketing order for domestic pistachios as established at 69 FR 17844 (April 5, 2004) and amended at 74 FR 56532 (November 2, 2009). Under the new regulation, ``pistachio'' means the nut of the... testing procedures that align with the Codex Alimentarius Commission's (Codex) sampling plan (75 FR...

  9. 76 FR 71465 - Defense Federal Acquisition Regulations Supplement; Notification Requirements for Awards of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-18

    ... Contracts (DFARS Case 2009-D036) AGENCY: Defense Acquisition Regulations System, Department of Defense (DoD... Logistics) Defense Procurement and Acquisition Policy (DPAP) Contract Policy and International Contracting... the senior procurement executive. (ii) The head of the agency must notify the congressional...

  10. Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation.

    PubMed

    Lord, Caleb C; Ferguson, Daniel; Thomas, Gwynneth; Brown, Amanda L; Schugar, Rebecca C; Burrows, Amy; Gromovsky, Anthony D; Betters, Jenna; Neumann, Chase; Sacks, Jessica; Marshall, Stephanie; Watts, Russell; Schweiger, Martina; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Lathia, Justin D; Sakaguchi, Takuya F; Lehner, Richard; Haemmerle, Guenter; Zechner, Rudolf; Brown, J Mark

    2016-07-26

    Adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes. PMID:27396333

  11. 75 FR 81337 - Interim Final Regulation Deferring the Reporting Date for Certain Data Elements Required Under...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-27

    ... of Federal Regulations EPA U.S. Environmental Protection Agency FR Federal Register GHG greenhouse... FR 56371, October 30, 2009). G. Executive Order 13045: Protection of Children From Environmental... From the Federal Register Online via the Government Publishing Office ] Part II...

  12. NOVA2-mediated RNA regulation is required for axonal pathfinding during development

    PubMed Central

    Saito, Yuhki; Miranda-Rottmann, Soledad; Ruggiu, Matteo; Park, Christopher Y; Fak, John J; Zhong, Ru; Duncan, Jeremy S; Fabella, Brian A; Junge, Harald J; Chen, Zhe; Araya, Roberto; Fritzsch, Bernd; Hudspeth, A J; Darnell, Robert B

    2016-01-01

    The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo. DOI: http://dx.doi.org/10.7554/eLife.14371.001 PMID:27223325

  13. 27 CFR 70.411 - Imposition of taxes, qualification requirements, and regulations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...: (1) Establishment and operation of distilled spirits plants. Part 19 of title 27 CFR contains the... vaporizing process. (2) Miscellaneous liquor transactions. Part 29 of 27 CFR contains miscellaneous... 27 CFR contains the regulations that. prescribe the gauging instruments, and methods or techniques...

  14. 75 FR 69423 - Information Collection Requirements; Defense Federal Acquisition Regulation Supplement; Small...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-12

    ... Control Number 0704-0386. Needs and Uses: DoD uses this information in assessing contractor compliance... Regulations System, Department of Defense (DoD). ACTION: Notice and request for comments regarding a proposed...)(A) of the Paperwork Reduction Act of 1995 (44 U.S.C. chapter 35), DoD announces the...

  15. 77 FR 74177 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Production...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-13

    ... Acquisition Regulations System, Department of Defense (DoD). ACTION: Notice; request for comments regarding a... 3506(c)(2)(A) of the Paperwork Reduction Act of 1995 (44 U.S.C. Chapter 35), DoD announces the proposed.... DoD invites comments on: (a) Whether the proposed collection of information is necessary for...

  16. 75 FR 6185 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Rights in...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-08

    ... Regulation Supplement; Rights in Technical Data and Computer Software (OMB Control Number 0704-0369) AGENCY... Subpart 227.72, Rights in Computer Software and Computer Software Documentation, and related provisions... technical data and computer software. DoD needs this information to implement 10 U.S.C. 2320, Rights...

  17. 75 FR 68330 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-05

    ... Regulation Supplement; Occupational Safety and Drug-Free Work Force (OMB Control Number 0704-0272) AGENCY... 223, Occupational Safety and Drug-Free Work Force, and related clauses in DFARS 252.223; OMB Control...; Identify the place of performance of all ammunition and explosives work; and Ensure contractor...

  18. 75 FR 3617 - Outer Continental Shelf Air Regulations Update To Include New Jersey State Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-22

    ... degrees longitude. \\1\\ The reader may refer to the Proposed Rulemaking, December 5, 1991 (56 FR 63774), and the preamble to the final rule promulgated September 4, 1992 (57 FR 40792) for further background and information on the OCS regulations. On October 2, 2009 (74 FR 50939), EPA proposed to...

  19. 78 FR 69267 - Defense Federal Acquisition Regulation Supplement: Requirements Relating to Supply Chain Risk...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-18

    ...DoD is issuing an interim rule amending the Defense Federal Acquisition Regulation Supplement (DFARS) to implement a section of the National Defense Authorization Act (NDAA) for Fiscal Year (FY) 2011, as amended by the NDAA for FY 2013. This interim rule allows DoD to consider the impact of supply chain risk in specified types of procurements related to national security...

  20. Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires β-catenin

    PubMed Central

    Parakh, Tehnaz N.; Hernandez, Jennifer A.; Grammer, Jean C.; Weck, Jennifer; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.; Nilson, John H.

    2006-01-01

    Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for β-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by β-catenin. Additionally, β-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of β-catenin. The stimulatory effect of β-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that β-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for β-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation. PMID:16895991

  1. ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary

    PubMed Central

    Pearson, John R.; Zurita, Federico; Tomás-Gallardo, Laura; Díaz-Torres, Alfonsa; Díaz de la Loza, María del Carmen; Franze, Kristian; Martín-Bermudo, María D.; González-Reyes, Acaimo

    2016-01-01

    The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis. PMID:26808525

  2. Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

    PubMed Central

    Weiss, S L; Sunde, R A

    1998-01-01

    Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3

  3. 76 FR 24443 - Federal Acquisition Regulation; Service Contracts Reporting Requirements; Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-02

    ... (202) 501-0044. Please cite FAR Case 2010-010. Correction In the proposed rule FR Doc. 2011-9515... simplified acquisition threshold (SAT). Contractors will be required to report on new fixed-price...

  4. Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

    PubMed Central

    Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

    2012-01-01

    Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

  5. Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

    PubMed Central

    Wendt, Michael K.; Taylor, Molly A.; Schiemann, Barbara J.; Schiemann, William P.

    2011-01-01

    Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth. PMID:21613543

  6. Up-regulation of glycolytic metabolism is required for HIF1α-driven bone formation.

    PubMed

    Regan, Jenna N; Lim, Joohyun; Shi, Yu; Joeng, Kyu Sang; Arbeit, Jeffrey M; Shohet, Ralph V; Long, Fanxin

    2014-06-10

    The bone marrow environment is among the most hypoxic in the body, but how hypoxia affects bone formation is not known. Because low oxygen tension stabilizes hypoxia-inducible factor alpha (HIFα) proteins, we have investigated the effect of expressing a stabilized form of HIF1α in osteoblast precursors. Brief stabilization of HIF1α in SP7-positive cells in postnatal mice dramatically stimulated cancellous bone formation via marked expansion of the osteoblast population. Remarkably, concomitant deletion of vascular endothelial growth factor A (VEGFA) in the mouse did not diminish bone accrual caused by HIF1α stabilization. Thus, HIF1α-driven bone formation is independent of VEGFA up-regulation and increased angiogenesis. On the other hand, HIF1α stabilization stimulated glycolysis in bone through up-regulation of key glycolytic enzymes including pyruvate dehydrogenase kinase 1 (PDK1). Pharmacological inhibition of PDK1 completely reversed HIF1α-driven bone formation in vivo. Thus, HIF1α stimulates osteoblast formation through direct activation of glycolysis, and alterations in cellular metabolism may be a broadly applicable mechanism for regulating cell differentiation. PMID:24912186

  7. UVRAG is required for organ rotation by regulating Notch endocytosis in Drosophila

    PubMed Central

    Lee, Gina; Liang, Chengyu; Park, Gihyun; Jang, Cholsoon; Jung, Jae U.; Chung, Jongkyeong

    2015-01-01

    Heterotaxy characterized by abnormal left–right body asymmetry causes diverse congenital anomalies. Organ rotation is a crucial developmental process to establish the left–right patterning during animal development. However, the molecular basis of how organ rotation is regulated is poorly understood. Here we report that Drosophila UV-resistance associated gene (UVRAG), a tumor suppressor that regulates autophagy and endocytosis, plays unexpected roles in controlling organ rotation. Loss-of-function mutants of UVRAG show seriously impaired organ rotation phenotypes, which are associated with defects in endocytic trafficking rather than autophagy. Blunted endocytic degradation by UVRAG deficiency causes endosomal accumulation of Notch, resulting in abnormally enhanced Notch activity. Knockdown of Notch itself or expression of a dominant negative form of Notch transcriptional co-activator Mastermind is sufficient to rescue the rotation defect in UVRAG mutants. Consistently, UVRAG-mutated heterotaxy patient cells also display highly increased Notch protein levels. These results suggest evolutionarily conserved roles of UVRAG in organ rotation by regulating Notch endocytic degradation. PMID:21729695

  8. Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Matukonis, Meghan K.; Bates, Paul

    2009-01-01

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. PMID:19013626

  9. Regulation of Motility Behavior in Myxococcus xanthus May Require an Extracytoplasmic-Function Sigma Factor

    PubMed Central

    Ward, Mandy J.; Lew, Helen; Treuner-Lange, Anke; Zusman, David R.

    1998-01-01

    Using interaction trap technology, we identified a putative extracytoplasmic-function (ECF) sigma factor (RpoE1) in Myxococcus xanthus, a bacterium which has a complex life cycle that includes fruiting body formation. The first domain of the response regulator protein FrzZ, a component of the Frz signal transduction system, was used as bait. Although the RpoE1 protein displayed no interactions with control proteins presented as bait, a weak interaction with a second M. xanthus response regulator (AsgA) was observed. While the specificity of the FrzZ-RpoE1 interaction therefore remains speculative, cloning and sequencing of the region surrounding rpoE1 localized it to a position downstream of the frzZ gene. A potential promoter site for binding of an ECF sigma factor was identified upstream of rpoE1, suggesting the gene may be autoregulated. However, primer extension studies suggested that transcription of rpoE1 occurs under both vegetative and developmental conditions from a ς70-like promoter. Dot blot analysis of RNA preparations confirmed the low-level, constitutive expression of rpoE1 during both stages of the life cycle. Analysis of an insertion mutant also indicated a role for RpoE1 under both vegetative and developmental conditions, since swarming was reduced on nutrient-rich agar and developmental aggregation was effected under starvation conditions, especially at high cell densities. An insertion mutation introduced into the gene directly downstream of rpoE1 (orf5) did not result in either swarming or developmental aggregation defects, even though the gene is transcribed as part of the same operon. Therefore, we propose that this new ECF sigma factor could play a role in the transcriptional regulation of genes involved in motility behavior during both stages of the complex M. xanthus life cycle. PMID:9791117

  10. Requirements for Ion and Solute Transport, and pH Regulation During Enamel Maturation

    PubMed Central

    LACRUZ, RODRIGO S.; SMITH, CHARLES E.; MOFFATT, PIERRE; CHANG, EUGENE H.; BROMAGE, TIMOTHY G.; BRINGAS, PABLO; NANCI, ANTONIO; BANIWAL, SANJEEV K.; ZABNER, JOSEPH; WELSH, MICHAEL J.; KURTZ, IRA; PAINE, MICHAEL L.

    2012-01-01

    Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, amutant porcine(CFTR-ΔF508) and the NBCe1-null mouse.Our data show that two SLCs(AE2 and NBCe1),Cftr,and Car2, Car3,Car6,and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of Cftr-ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. PMID:21732355

  11. Requirements for ion and solute transport, and pH regulation during enamel maturation.

    PubMed

    Lacruz, Rodrigo S; Smith, Charles E; Moffatt, Pierre; Chang, Eugene H; Bromage, Timothy G; Bringas, Pablo; Nanci, Antonio; Baniwal, Sanjeev K; Zabner, Joseph; Welsh, Michael J; Kurtz, Ira; Paine, Michael L

    2012-04-01

    Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR-ΔF508) and the NBCe1-null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR-ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. PMID:21732355

  12. Leptin is required for hypothalamic regulation of miRNAs targeting POMC 3′UTR

    PubMed Central

    Derghal, Adel; Djelloul, Mehdi; Airault, Coraline; Pierre, Clément; Dallaporta, Michel; Troadec, Jean-Denis; Tillement, Vanessa; Tardivel, Catherine; Bariohay, Bruno; Trouslard, Jérôme; Mounien, Lourdes

    2015-01-01

    The central nervous system (CNS) monitors modifications in metabolic parameters or hormone levels and elicits adaptive responses such as food intake regulation. Particularly, within the hypothalamus, leptin modulates the activity of pro-opiomelanocortin (POMC) neurons which are critical regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the POMC gene causes hyperphagia and obesity. MicroRNAs (miRNAs) are short noncoding RNA molecules that post-transcriptionally repress the expression of genes by binding to 3′-untranslated regions (3′UTR) of the target mRNAs. However, little is known regarding the role of miRNAs that target POMC 3′UTR in the central control energy homeostasis. Particularly, their interaction with the leptin signaling pathway remain unclear. First, we used common prediction programs to search for potential miRNAs target sites on 3′UTR of POMC mRNA. This screening identified a set of conserved miRNAs seed sequences for mir-383, mir-384-3p, and mir-488. We observed that mir-383, mir-384-3p, and mir-488 are up-regulated in the hypothalamus of leptin deficient ob/ob mice. In accordance with these observations, we also showed that mir-383, mir-384-3p, and mir-488 were increased in db/db mice that exhibit a non-functional leptin receptor. The intraperitoneal injection of leptin down-regulated the expression of these miRNAs of interest in the hypothalamus of ob/ob mice showing the involvement of leptin in the expression of mir-383, mir-384-3p, and mir-488. Finally, the evaluation of responsivity to intracerebroventricular administration of leptin exhibited that a chronic treatment with leptin decreased mir-488 expression in hypothalamus of C57BL/6 mice. In summary, these results suggest that leptin modulates the expression of miRNAs that target POMC mRNA in hypothalamus. PMID:25999818

  13. FOXO1 is Required for Binding of PR on IRF4, Novel Transcriptional Regulator of Endometrial Stromal Decidualization

    PubMed Central

    Vasquez, Yasmin M.; Mazur, Erik C.; Li, Xilong; Kommagani, Ramakrishna; Jiang, Lichun; Chen, Rui; Lanz, Rainer B.; Kovanci, Ertug; Gibbons, William E.

    2015-01-01

    The forkhead box O1A (FOXO1) is an early-induced target of the protein kinase A pathway during the decidualization of human endometrial stromal cells (HESCs). In this study we identified the cistrome and transcriptome of FOXO1 and its role as a transcriptional regulator of the progesterone receptor (PR). Direct targets of FOXO1 were identified by integrating RNA sequencing with chromatin immunoprecipitation followed by deep sequencing. Gene ontology analysis demonstrated that FOXO1 regulates a subset of genes in decidualization such as those involved in cancer, p53 signaling, focal adhesions, and Wnt signaling. An overlap of the FOXO1 and PR chromatin immunoprecipitation followed by deep sequencing intervals revealed the co-occupancy of FOXO1 in more than 75% of PR binding intervals. Among these intervals were highly enriched motifs for the interferon regulatory factor member 4 (IRF4). IRF4 was determined to be a genomic target of both FOXO1 and PR and also to be differentially regulated in HESCs treated with small interfering RNA targeting FOXO1 or PR prior to decidualization stimulus. Ablation of FOXO1 was found to abolish binding of PR to the shared binding interval downstream of the IRF4 gene. Finally, small interfering RNA-mediated ablation of IRF4 was shown to compromise morphological transformation of decidualized HESCs and to attenuate the expression of the decidual markers IGFBP1, PRL, and WNT4. These results provide the first evidence that FOXO1 is functionally required for the binding of PR to genomic targets. Most notably, FOXO1 and PR are required for the regulation of IRF4, a novel transcriptional regulator of decidualization in HESCs. PMID:25584414

  14. A Mechanism Regulating G Protein-coupled Receptor Signaling That Requires Cycles of Protein Palmitoylation and Depalmitoylation* ♦

    PubMed Central

    Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H.; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M.; Martin, Brent R.; Mennerick, Steven J.; Blumer, Kendall J.

    2014-01-01

    Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K+ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders. PMID:24385443

  15. 77 FR 16196 - Delaying the Compliance Date for Certain Requirements of the Regulations Implementing Titles II...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-20

    ...By this rule, the Department of Justice is proposing to extend the date for compliance with certain requirements in the 2010 Americans with Disabilities Act (ADA) Standards for Accessible Design that relate to provision of accessible entry and exit for existing swimming pools and spas. Concurrently with the publication of this Notice of Proposed Rulemaking (NPRM), the Department is publishing......

  16. 75 FR 34277 - Federal Acquisition Regulation; FAR Case 2008-007, Additional Requirements for Market Research

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ... Research AGENCIES: Department of Defense (DoD), General Services Administration (GSA), and National... executive agencies. Specifically, the head of the agency must conduct market research before issuing an... million for the procurement of items other than commercial items is required to conduct market...

  17. 75 FR 40716 - Defense Federal Acquisition Regulation Supplement; Notification Requirements for Awards of Single...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-13

    ...; Notification Requirements for Awards of Single-Source Task or Delivery Orders (DFARS Case 2009-D036) AGENCY...-Source Task or Delivery Orders.'' 10 U.S.C. 2304a(d)(3)(A) prohibits the award of a sole-source task or... agency determines in writing that-- (1) The task or delivery orders expected under the contract are...

  18. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... established under § 195.406 corresponding to— (i) A stress level greater than 20-percent of the specified minimum yield strength of the line pipe; or (ii) If the stress level is unknown or the pipeline is not... subpart H of this part, except corrosion control is not required for pipelines existing on July 3,...

  19. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... established under § 195.406 corresponding to— (i) A stress level greater than 20-percent of the specified minimum yield strength of the line pipe; or (ii) If the stress level is unknown or the pipeline is not... subpart H of this part, except corrosion control is not required for pipelines existing on July 3,...

  20. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... established under § 195.406 corresponding to— (i) A stress level greater than 20-percent of the specified minimum yield strength of the line pipe; or (ii) If the stress level is unknown or the pipeline is not... subpart H of this part, except corrosion control is not required for pipelines existing on July 3,...

  1. 49 CFR 195.11 - What is a regulated rural gathering line and what requirements apply?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... established under § 195.406 corresponding to— (i) A stress level greater than 20-percent of the specified minimum yield strength of the line pipe; or (ii) If the stress level is unknown or the pipeline is not... subpart H of this part, except corrosion control is not required for pipelines existing on July 3,...

  2. 75 FR 52622 - Amendment to the International Traffic in Arms Regulations: Removing Requirement for Prior...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-27

    ... armed forces of a foreign country (42 FR 41631, dated August 18, 1977). Also, Sec. 124.06, entitled... effective January 1, 1985 (49 FR 47682, dated December 6, 1984). Section 126.8 did not require prior... approval, was added to Sec. 126.8 in a final rule effective March 31, 1985 (50 FR 12787, dated April...

  3. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false What general requirements apply to pipelines... (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) PIPELINE SAFETY TRANSPORTATION OF NATURAL AND OTHER GAS BY PIPELINE: MINIMUM FEDERAL SAFETY...

  4. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 false What general requirements apply to pipelines... (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) PIPELINE SAFETY TRANSPORTATION OF NATURAL AND OTHER GAS BY PIPELINE: MINIMUM FEDERAL SAFETY...

  5. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false What general requirements apply to pipelines... (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) PIPELINE SAFETY TRANSPORTATION OF NATURAL AND OTHER GAS BY PIPELINE: MINIMUM FEDERAL SAFETY...

  6. 76 FR 64264 - Authority to Require Supervision and Regulation of Certain Nonbank Financial Companies

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-18

    ... comment on a proposed rule that would establish these requirements. See 76 FR 7731 (February 11, 2011... advance notice of proposed rulemaking (the ``ANPR'') on October 6, 2010 (75 FR 61653), in which it... notice of proposed rulemaking (the ``NPR'') (76 FR 4555) through which it sought public comment...

  7. 75 FR 61653 - Advance Notice of Proposed Rulemaking Regarding Authority To Require Supervision and Regulation...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-06

    ...Section 113 of the Dodd-Frank Wall Street Reform and Consumer Protection Act (the ``DFA'') gives the Financial Stability Oversight Council (the ``Council'') the authority to require that a nonbank financial company be supervised by the Board of Governors of the Federal Reserve System (``Board of Governors'') and subject to prudential standards if the Council determines that material financial......

  8. 49 CFR 192.13 - What general requirements apply to pipelines regulated under this part?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false What general requirements apply to pipelines... (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) PIPELINE SAFETY TRANSPORTATION OF NATURAL AND OTHER GAS BY PIPELINE: MINIMUM FEDERAL SAFETY...

  9. 78 FR 60216 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-01

    ... DHS Department of Homeland Security FR Federal Register NPRM Notice of Proposed Rulemaking A... requirements for a two-year period. This suspension was published in the Federal Register at 76 FR 1360... this suspension until midnight, September 30, 2013 (78 FR 25). At this time, there is no plan to...

  10. 78 FR 25 - Regulated Navigation Area; Reporting Requirements for Barges Loaded With Certain Dangerous...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-02

    ... stay was previously published at 76 Federal Register (FR) 1360 (January 10, 2011), which expires... Acronyms CDC Certain Dangerous Cargo DHS Department of Homeland Security FR Federal Register NPRM Notice of... requirements for a two-year period. This suspension was published in the Federal Register at 76 FR...

  11. 75 FR 60254 - Federal Acquisition Regulation; Certification Requirement and Procurement Prohibition Relating to...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-29

    ... (SAT), contracts for the acquisition of commercial items, and contracts for the acquisition of... the SAT. It is intended to limit the applicability of laws to them. FASA provides that if a provision... below the SAT, the law will apply to them. Therefore, given that the requirements of sections 102...

  12. 77 FR 8757 - Revising Underground Storage Tank Regulations-Revisions to Existing Requirements and New...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-15

    ... Register on November 18, 2011 (76 FR 71708) (FRL-9485-5). These changes establish federal requirements that... health and the environment by increasing the number of prevented UST releases and quickly detecting them...., Washington, DC. The Public Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday through Friday,...

  13. 77 FR 66464 - Federal Acquisition Regulation; Submission for OMB Review; Value Engineering Requirements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-05

    ... published in the Federal Register at 77 FR 43076, on July 23, 2012. No comments were received. Public... Register at 77 FR 43076, on July 23, 2012. Respondents: 1,934. Responses per Respondent: 2. Annual...., Washington, DC 20417. ATTN: Hada Flowers/IC 9000-0027, Value Engineering Requirements. Instructions:...

  14. 76 FR 53886 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-30

    ... 252.217-7026, Identification of Sources of Supply; and 252.217- 7028, Over and Above Work; OMB Control... it limits the Government's liability as a related party to the work the contractor performs... above'' work before the work commences. This requirement allows the Government to review the need...

  15. 76 FR 14548 - Federal Acquisition Regulation; Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... establishing a BPA that exceeds the SAT (see FAR 8.405-4); and Adds language explaining that the protest..., Ordering procedures for supplies, and services not requiring a statement of work. Current language at FAR 8... process under which the FSS Program operates. For example, section 863 uses language such as...

  16. 78 FR 27908 - Federal Management Regulation (FMR); Mail Management; Financial Requirements for All Agencies

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-13

    .... Background On June 6, 2002, GSA published an interim rule in the Federal Register (67 FR 38899) that required..., 2003 (68 FR 56112). Many agencies unfortunately were not able to meet this goal. If agencies did not..., published in the Federal Register on May 13, 2008 (73 FR 27540), granted a 12 month deviation from April...

  17. Beyond Transcription Factors: The Role of Chromatin Modifying Enzymes in Regulating Transcription Required for Memory

    ERIC Educational Resources Information Center

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately…

  18. Extracellular signal-regulated kinase (ERK) activation is required for porcine epidemic diarrhea virus replication.

    PubMed

    Kim, Youngnam; Lee, Changhee

    2015-10-01

    Porcine epidemic diarrhea virus (PEDV) is a highly enteropathogenic coronavirus of swine that causes acute enteritis with high mortality in nursery piglets. To date, the cellular factors involved in PEDV replication have not been well defined. The extracellular signal-regulated kinase (ERK) that serves as a critical component of cellular signal transduction pathways to modulate a variety of cellular functions has been shown to regulate several viral infections. In the present study, we found that PEDV activates ERK1/2 early in infection independently of viral replication. The PEDV-induced ERK1/2 activation resulted in the phosphorylation of its downstream substrate Elk-1 in infected cells. Treatment with ERK inhibitors or ERK1/2 knockdown significantly suppressed viral progeny production. Inhibition of ERK activation also diminished viral protein expression and genomic and subgenomic RNA transcription. These findings indicate that the ERK signaling pathway plays an important role in the PEDV life cycle and beneficially contributes to viral infection. PMID:26115165

  19. Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA

    PubMed Central

    Brimacombe, Cedric A.; Ding, Hao; Johnson, Jeanette A.

    2015-01-01

    ABSTRACT Gene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCE Horizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of

  20. Collagen I and the fibroblast: high protein expression requires a new paradigm of post-transcriptional, feedback regulation

    PubMed Central

    Schwarz, Richard I.

    2016-01-01

    Background Scaling protein production seems like a simple perturbation of transcriptional control. However, when embryonic tendon fibroblasts have to produce >50% procollagen and secrete it from the cell 4 times faster than the average protein, this taxes the cellular machinery and requires a fresh look at how the pathway is controlled. Ascorbate, a reducing agent, can stimulate procollagen production 6-fold. Procollagen mRNA levels goes up 6-fold but requires 3 days for the cell to accomplish this task. Secretion rates, the last cellular step in the process, also goes up 6-fold but this occurs in <1 h. What regulatory scheme is consistent with these properties? Scope of this review This review focuses on fibroblasts that make high levels of procollagen (type I) and how they regulate the collagen pathway. Data from many different labs are relevant to this problem but it is hard to see the bigger picture from a large number of small studies. This review aims to consolidate this data into a coherent model and this requires solutions to some controversies and postulating potential mechanisms where the details are still missing. Major Conclusions In high collagen producing cells, the pathway is controlled by post-transcriptional regulation. This requires feedback control between secretion and translation rates that is based on the helical structure of the procollagen molecule and additional tissue-specific modifications. General Significance Transcriptional control does not scale well to high protein production with rapid regulation. New paradigms lead to better understanding of collagen diseases and tendon morphogenesis. PMID:26900604

  1. The Ustilago maydis Nit2 Homolog Regulates Nitrogen Utilization and Is Required for Efficient Induction of Filamentous Growth

    PubMed Central

    Horst, Robin J.; Zeh, Christine; Saur, Alexandra; Sonnewald, Sophia; Sonnewald, Uwe

    2012-01-01

    Nitrogen catabolite repression (NCR) is a regulatory strategy found in microorganisms that restricts the utilization of complex and unfavored nitrogen sources in the presence of favored nitrogen sources. In fungi, this concept has been best studied in yeasts and filamentous ascomycetes, where the GATA transcription factors Gln3p and Gat1p (in yeasts) and Nit2/AreA (in ascomycetes) constitute the main positive regulators of NCR. The reason why functional Nit2 homologs of some phytopathogenic fungi are required for full virulence in their hosts has remained elusive. We have identified the Nit2 homolog in the basidiomycetous phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, positive regulator of nitrogen utilization. By transcriptome analysis of sporidia grown on artificial media devoid of favored nitrogen sources, we show that only a subset of nitrogen-responsive genes are regulated by Nit2, including the Gal4-like transcription factor Ton1 (a target of Nit2). Ustilagic acid biosynthesis is not under the control of Nit2, while nitrogen starvation-induced filamentous growth is largely dependent on functional Nit2. nit2 deletion mutants show the delayed initiation of filamentous growth on maize leaves and exhibit strongly compromised virulence, demonstrating that Nit2 is required to efficiently initiate the pathogenicity program of U. maydis. PMID:22247264

  2. The Ustilago maydis Nit2 homolog regulates nitrogen utilization and is required for efficient induction of filamentous growth.

    PubMed

    Horst, Robin J; Zeh, Christine; Saur, Alexandra; Sonnewald, Sophia; Sonnewald, Uwe; Voll, Lars M

    2012-03-01

    Nitrogen catabolite repression (NCR) is a regulatory strategy found in microorganisms that restricts the utilization of complex and unfavored nitrogen sources in the presence of favored nitrogen sources. In fungi, this concept has been best studied in yeasts and filamentous ascomycetes, where the GATA transcription factors Gln3p and Gat1p (in yeasts) and Nit2/AreA (in ascomycetes) constitute the main positive regulators of NCR. The reason why functional Nit2 homologs of some phytopathogenic fungi are required for full virulence in their hosts has remained elusive. We have identified the Nit2 homolog in the basidiomycetous phytopathogen Ustilago maydis and show that it is a major, but not the exclusive, positive regulator of nitrogen utilization. By transcriptome analysis of sporidia grown on artificial media devoid of favored nitrogen sources, we show that only a subset of nitrogen-responsive genes are regulated by Nit2, including the Gal4-like transcription factor Ton1 (a target of Nit2). Ustilagic acid biosynthesis is not under the control of Nit2, while nitrogen starvation-induced filamentous growth is largely dependent on functional Nit2. nit2 deletion mutants show the delayed initiation of filamentous growth on maize leaves and exhibit strongly compromised virulence, demonstrating that Nit2 is required to efficiently initiate the pathogenicity program of U. maydis. PMID:22247264

  3. Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution

    PubMed Central

    Lee, Wook-Bin; Kang, Ji-Seon; Choi, Won Young; Zhang, Quanri; Kim, Chul Han; Choi, Un Yung; Kim-Ha, Jeongsil; Kim, Young-Joon

    2016-01-01

    In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls. PMID:27089465

  4. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or... exemption. (3) Division 2.3 “poisonous gas”, as listed in 49 CFR 172.101 that is also a “material...

  5. 33 CFR 165.830 - Regulated Navigation Area; Reporting Requirements for Barges Loaded with Certain Dangerous...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...— Barge means a non-self propelled vessel engaged in commerce, as set out in 33 CFR 160.204. Certain... CFR 173.50. (2) Division 1.5D blasting agents for which a permit is required under 49 CFR 176.415 or... exemption. (3) Division 2.3 “poisonous gas”, as listed in 49 CFR 172.101 that is also a “material...

  6. 75 FR 33271 - Information Collection Requirement; Defense Federal Acquisition Regulation Supplement; Foreign...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-11

    ...In compliance with Section 3506(c)(2)(A) of the Paperwork Reduction Act of 1995 (44 U.S.C. Chapter 35), DoD announces the proposed extension of a public information collection requirement and seeks public comment on the provisions thereof. DoD invites comments on: (a) Whether the proposed collection of information is necessary for the proper performance of the functions of DoD, including......

  7. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi

    PubMed Central

    Zuber, Johannes; McJunkin, Katherine; Fellmann, Christof; Dow, Lukas E; Taylor, Meredith J; Hannon, Gregory J; Lowe, Scott W

    2012-01-01

    Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo1. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets. PMID:21131983

  8. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  9. Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe

    PubMed Central

    Suárez, M Belén; Alonso-Nuñez, María Luisa; del Rey, Francisco; McInerny, Christopher J; Vázquez de Aldana, Carlos R

    2015-01-01

    The division cycle of unicellular yeasts is completed with the activation of a cell separation program that results in the dissolution of the septum assembled during cytokinesis between the 2 daughter cells, allowing them to become independent entities. Expression of the eng1+ and agn1+ genes, encoding the hydrolytic enzymes responsible for septum degradation, is activated at the end of each cell cycle by the transcription factor Ace2. Periodic ace2+ expression is regulated by the transcriptional complex PBF (PCB Binding Factor), composed of the forkhead-like proteins Sep1 and Fkh2 and the MADS box-like protein Mbx1. In this report, we show that Ace2-dependent genes contain several combinations of motifs for Ace2 and PBF binding in their promoters. Thus, Ace2, Fkh2 and Sep1 were found to bind in vivo to the eng1+ promoter. Ace2 binding was coincident with maximum level of eng1+ expression, whereas Fkh2 binding was maximal when mRNA levels were low, supporting the notion that they play opposing roles. In addition, we found that the expression of eng1+ and agn1+ was differentially affected by mutations in PBF components. Interestingly, agn1+ was a major target of Mbx1, since its ectopic expression resulted in the suppression of Mbx1 deletion phenotypes. Our results reveal a complex regulation system through which the transcription factors Ace2, Fkh2, Sep1 and Mbx1 in combination control the expression of the genes involved in separation at the end of the cell division cycle. PMID:26237280

  10. Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe.

    PubMed

    Suárez, M Belén; Alonso-Nuñez, María Luisa; del Rey, Francisco; McInerny, Christopher J; Vázquez de Aldana, Carlos R

    2015-01-01

    The division cycle of unicellular yeasts is completed with the activation of a cell separation program that results in the dissolution of the septum assembled during cytokinesis between the 2 daughter cells, allowing them to become independent entities. Expression of the eng1(+) and agn1(+) genes, encoding the hydrolytic enzymes responsible for septum degradation, is activated at the end of each cell cycle by the transcription factor Ace2. Periodic ace2(+) expression is regulated by the transcriptional complex PBF (PCB Binding Factor), composed of the forkhead-like proteins Sep1 and Fkh2 and the MADS box-like protein Mbx1. In this report, we show that Ace2-dependent genes contain several combinations of motifs for Ace2 and PBF binding in their promoters. Thus, Ace2, Fkh2 and Sep1 were found to bind in vivo to the eng1(+) promoter. Ace2 binding was coincident with maximum level of eng1(+) expression, whereas Fkh2 binding was maximal when mRNA levels were low, supporting the notion that they play opposing roles. In addition, we found that the expression of eng1(+) and agn1(+) was differentially affected by mutations in PBF components. Interestingly, agn1(+) was a major target of Mbx1, since its ectopic expression resulted in the suppression of Mbx1 deletion phenotypes. Our results reveal a complex regulation system through which the transcription factors Ace2, Fkh2, Sep1 and Mbx1 in combination control the expression of the genes involved in separation at the end of the cell division cycle. PMID:26237280

  11. PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp.

    PubMed Central

    van Velkinburgh, Jennifer C.; Gunn, John S.

    1999-01-01

    As enteric pathogens, Salmonella spp. are resistant to the actions of bile. Salmonella typhimurium and Salmonella typhi strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-type S. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% bile dramatically increased bile resistance. Mutant strains of S. typhimurium and S. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC and prgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder. PMID:10084994

  12. CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

    PubMed Central

    Wright, Roni H.G.; Castellano, Giancarlo; Bonet, Jaume; Le Dily, Francois; Font-Mateu, Jofre; Ballaré, Cecilia; Nacht, A. Silvina; Soronellas, Daniel; Oliva, Baldo; Beato, Miguel

    2012-01-01

    Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer. PMID:22948662

  13. A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo.

    PubMed

    Brenner, Daniel S; Golden, Judith P; Vogt, Sherri K; Dhaka, Ajay; Story, Gina M; Gereau Iv, Robert W

    2014-10-01

    The ability to sense and respond to thermal stimuli at varied environmental temperatures is essential for survival in seasonal areas. In this study, we show that mice respond similarly to ramping changes in temperature from a wide range of baseline temperatures. Further investigation suggests that this ability to adapt to different ambient environments is based on rapid adjustments made to a dynamic temperature set point. The adjustment of this set point requires transient receptor potential cation channel, subfamily member 8 (TRPM8), but not transient receptor potential cation channel, subfamily A, member 1 (TRPA1), and is regulated by phospholipase C (PLC) activity. Overall, our findings suggest that temperature response thresholds in mice are dynamic, and that this ability to adapt to environmental temperature seems to mirror the in vitro findings that PLC-mediated hydrolysis of phosphoinositol 4,5-bisphosphate modulates TRPM8 activity and thereby regulates the response thresholds to cold stimuli. PMID:25109670

  14. Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis.

    PubMed

    Gray, Lawrence R; Sultana, Mst Rasheda; Rauckhorst, Adam J; Oonthonpan, Lalita; Tompkins, Sean C; Sharma, Arpit; Fu, Xiaorong; Miao, Ren; Pewa, Alvin D; Brown, Kathryn S; Lane, Erin E; Dohlman, Ashley; Zepeda-Orozco, Diana; Xie, Jianxin; Rutter, Jared; Norris, Andrew W; Cox, James E; Burgess, Shawn C; Potthoff, Matthew J; Taylor, Eric B

    2015-10-01

    Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D. PMID:26344103

  15. Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

    PubMed Central

    Bartek, I. L.; Woolhiser, L. K.; Baughn, A. D.; Basaraba, R. J.; Jacobs, W. R.; Lenaerts, A. J.

    2014-01-01

    ABSTRACT To survive a dynamic host environment, Mycobacterium tuberculosis must endure a series of challenges, from reactive oxygen and nitrogen stress to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology of M. tuberculosis, we generated a strain deleted for lsr2. Analysis of the M. tuberculosis Δlsr2 strain demonstrated that Lsr2 is not required for DNA protection, as this strain was equally susceptible as the wild type to DNA-damaging agents. The lsr2 mutant did display severe growth defects under normoxic and hyperoxic conditions, but it was not required for growth under low-oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during anaerobiosis, as well as a lag in recovery from it. Gene expression profiling of the Δlsr2 mutant under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicates that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The Δlsr2 strain was capable of establishing an early infection in the BALB/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrate M. tuberculosis Lsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection. PMID:24895305

  16. Regulation of the ERK pathway in the dentate gyrus by in vivo dopamine D1 receptor stimulation requires glutamatergic transmission.

    PubMed

    Gangarossa, Giuseppe; Valjent, Emmanuel

    2012-11-01

    Acute systemic administration of the dopamine D1/D5 receptors (D1Rs) agonist, SKF81297, activates the extracellular signal-regulated protein kinases (ERK) pathway selectively in the granule cells of the dentate gyrus. In this study, we examined the mechanisms involved in this regulation and investigated the molecular components that could promote ERK-dependent transcription and translation. SKF81297 induced phosphorylation of ERK and histone H3 required intact glutamatergic transmission. Blockade of glutamate release achieved by the mGluR2/3 agonist, LY354740 or the selective adenosine A1R agonist, CCPA as well as neurotoxic lesions of lateral entorhinal cortex reduced the ability of SKF81297 to induce ERK activation in the dentate gyrus. This activation required the combined stimulation of NR2B-containing NMDARs, mGluR1 and mGluR5. SKF81297 evoked phosphorylation of the ribosomal protein S6 (rpS6) selectively at the Ser235/236 site while the Ser240/244 site remains unchanged. The SKF81297 induced increased phosphorylation of rpS6 was dependent on PKC and ERK/p90RSK activation. Surprisingly, administration of D1Rs agonist suppressed mTORC1/p70S6K pathway suggesting an mTOR-independent regulation of rpS6 phosphorylation. Taken together, our results show that intact glutamatergic transmission plays a major role in the regulation of ERK-dependent phosphorylation of histone H3 and rpS6 observed in the mouse dentate gyrus after systemic administration of SKF81297. PMID:22796106

  17. Eya4 regulation of Na+/K+-ATPase is required for sensory system development in zebrafish.

    PubMed

    Wang, Libin; Sewell, William F; Kim, Sang D; Shin, Jordan T; MacRae, Calum A; Zon, Leonard I; Seidman, J G; Seidman, Christine E

    2008-10-01

    To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, we studied eya4 expression during zebrafish development and characterized eya4 deficiency. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4 focused our analyses on atp1b2b, which encodes the beta2b subunit of the zebrafish Na+/K+-ATPase. We demonstrate atp1b2b levels are reduced in eya4 morphant fish and that morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. We conclude that eya4 regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish. PMID:18799547

  18. HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3

    PubMed Central

    Dilg, Daniel; Saleh, Rasha Noureldin M.; Phelps, Sarah Elizabeth Lee; Rose, Yoann; Dupays, Laurent; Murphy, Cian; Mohun, Timothy; Anderson, Robert H.; Scambler, Peter J.; Chapgier, Ariane L. A.

    2016-01-01

    Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor. PMID:27518902

  19. HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3.

    PubMed

    Dilg, Daniel; Saleh, Rasha Noureldin M; Phelps, Sarah Elizabeth Lee; Rose, Yoann; Dupays, Laurent; Murphy, Cian; Mohun, Timothy; Anderson, Robert H; Scambler, Peter J; Chapgier, Ariane L A

    2016-01-01

    Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor. PMID:27518902

  20. Meis1 Regulates Epidermal Stem Cells and Is Required for Skin Tumorigenesis

    PubMed Central

    Okumura, Kazuhiro; Saito, Megumi; Isogai, Eriko; Aoto, Yoshimasa; Hachiya, Tsuyoshi; Sakakibara, Yasubumi; Katsuragi, Yoshinori; Hirose, Satoshi; Kominami, Ryo; Goitsuka, Ryo; Nakamura, Takuro; Wakabayashi, Yuichi

    2014-01-01

    Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1. PMID:25013928

  1. Impacts & Compliance Implementation Plans & Required Deviations for Toxic Substance Control Act (TSCA) Regulation of Double Shell Tanks (DST)

    SciTech Connect

    MULKEY, C.H.

    2000-08-22

    In May 2000, the U.S. Department of Energy, Office of River Protection (DOE-ORP) and the U.S. Environmental Protection Agency (EPA) held meetings regarding the management of polychlorinated biphenyls (PCBs) in the Hanford tank waste. It was decided that the radioactive waste currently stored in the double-shell tanks (DSTs) contain waste which will become subject to the Toxic Substance Control Act (TSCA) (40 CFR 761). As a result, DOE-ORP directed the River Protection Project tank farm contractor (TFC) to prepare plans for managing the PCB inventory in the DSTs. Two components of the PCB management plans are this assessment of the operational impacts of TSCA regulation and the identifications of deviations from TSCA that are required to accommodate tank farm unique limitations. This plan provides ORP and CH2M HILL Hanford Group, Inc. (CHG) with an outline of TSCA PCB requirements and their applicability to tank farm activities, and recommends a compliance/implementation approach. Where strict compliance is not possible, the need for deviations from TSCA PCB requirements is identified. The purpose of assembling this information is to enhance the understanding of PCB management requirements, identify operational impacts and select impact mitigation strategies. This information should be useful in developing formal agreements with EPA where required.

  2. Using reporting requirements to improve employer wellness incentives and their regulation.

    PubMed

    Madison, Kristin; Schmidt, Harald; Volpp, Kevin G

    2014-10-01

    Employer interest in offering financial incentives for healthy behaviors has been increasing. Some employers have begun to tie health plan-based rewards or penalties to standards involving tobacco use or biometric measures such as body mass index. The Patient Protection and Affordable Care Act attempts to strike a balance between the potential benefits and risks of wellness incentive programs by permitting these incentives but simultaneously limiting their use. Evidence about the implications of the newest generation of incentive programs for health, health costs, and burdens on individual employees will be critical for informing both private and public decision makers. After describing the many pieces of information that would be valuable for assessing these programs, this article proposes more narrowly targeted reporting requirements that could facilitate incentive program development, evaluation, and oversight. PMID:25037837

  3. Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process.

    PubMed Central

    Drucker, H

    1975-01-01

    To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process. PMID:125263

  4. JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

    PubMed Central

    Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

    2006-01-01

    The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

  5. Strengthening Indonesia’s Field Epidemiology Training Programme to address International Health Regulations requirements

    PubMed Central

    Samaan, Gina; Santoso, Hari; Kushadiwijaya, Haripurnomo; Juwita, Ratna; Mohadir, Andi; Aditama, Tjandra

    2010-01-01

    Abstract Problem According to the International Health Regulations (IHR), countries need to strengthen core capacity for disease surveillance and response systems. Many countries are establishing or enhancing their field epidemiology training programmes (FETPs) to meet human resource needs but face challenges in sustainability and training quality. Indonesia is facing these challenges, which include limited resources for field training and limited coordination in a newly decentralized health system. Approach A national FETP workplan was developed based on an evaluation of the existing programme and projected human resource needs. A Ministry of Health Secretariat linking universities, national and international partners was established to oversee revision and implementation of the FETP. Local setting The FETP is integrated into the curriculum of Indonesian universities and field training is conducted in district and provincial health offices under the coordination of the universities and the FETP Secretariat. Relevant changes The FETP was included in the Ministry of Health workforce development strategy through governmental decree. Curricula have been enhanced and field placements strengthened to provide trainees with better learning experiences. To improve sustainability of the FETP, links were established with the Indonesian Epidemiologists’ Association, local governments and donors to cultivate future FETP champions and maintain funding. Courses, competitions and discussion forums were established for field supervisors and alumni. These changes have increased the geographic distribution of students, intersectoral and international participation and the quality of student performance. Lessons learnt The main lesson learnt is that linkages with universities, ministries and international agencies such as the World Health Organization are critical for building a sustainable high-quality programme. The most critical factors were development of trusting relationships

  6. Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation.

    PubMed

    Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Hartmann, Alain; Piveteau, Pascal

    2015-08-01

    In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a ΔagrA deletion mutant were compared in a collection of 10 soil microcosms. The ΔagrA mutant displayed significantly reduced survival in these biotic soil microcosms, and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional, while the variations in the transcriptomes between the wild type and the ΔagrA deletion mutant were modest under abiotic conditions. Indeed, in biotic soil environments, 578 protein-coding genes and an extensive repertoire of noncoding RNAs (ncRNAs) were differentially transcribed. The transcription of genes coding for proteins involved in cell envelope and cellular processes, including the phosphotransferase system and ABC transporters, and proteins involved in resistance to antimicrobial peptides was affected. Under sterilized soil conditions, the differences were limited to 86 genes and 29 ncRNAs. These results suggest that the response regulator AgrA of the Agr communication system plays important roles during the saprophytic life of L. monocytogenes in soil. PMID:26002901

  7. SRC-3 is required for CAR-regulated hepatocyte proliferation and drug metabolism

    PubMed Central

    Chen, Tenghui; Chen, Qiang; Xu, Yixiang; Zhou, Qiling; Zhu, Jingwei; Zhang, Hao; Wu, Qiao; Xu, Jianming; Yu, Chundong

    2011-01-01

    Background & Aims Nuclear receptors such as pregnane X receptor and constitutive androstane receptor (CAR) are important regulators of drug-metabolizing systems such as P450s enzymes and modulate xenobiotic metabolism as well as hepatocellular proliferation. Binding of CAR to NR response elements alone is not sufficient to activate gene expression. Here we investigate the role of steroid receptor coactivator (SRC) family members in CAR-mediated hepatocyte proliferation and drug metabolism. Methods The role of SRCs in CAR activation was assessed in cell-based transfection assays and protein-protein interaction assays. The in vivo role of SRCs in CAR-mediated hepatocyte proliferation and drug metabolism was examined by using mice deficient in SRCs. Results SRC-3 displayed the highest coactivating activity to CAR compared with SRC-1 and SRC-2 in a cell-based reporter assay. Knockout of SRC-3 in mice attenuated hepatic hyperplasia induced by a CAR agonist 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), which was associated with a reduced expression of c-Myc and Foxm-1. In contrast, knockout of SRC-1 or SRC-2 in mice did not affect TCPOBOP-induced hepatic hyperplasia. SRC-3-deficient mice were hypersensitive to zoxazolamine-induced paralysis, but were resistant to acetaminophen hepatotoxicity induced by TCPOBOP, whereas mutant mice deficient in SRC-1 or SRC-2 exhibited severe acetaminophen hepatotoxicity similar to wild-type controls. Accordingly, deficiency in SRC-3, but not SRC-1 or SRC-2, resulted in a reduced CAR-mediated expression of drug metabolism-related genes in the liver. Conclusions Our study demonstrates that SRC-3 is the predominant transcriptional coactivator among the three SRC family members for CAR activation to promote hepatocyte proliferation and drug metabolism. PMID:21827731

  8. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

    PubMed

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes. PMID:26741360

  9. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence

    PubMed Central

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W.

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes. PMID:26741360

  10. Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation

    PubMed Central

    Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Hartmann, Alain

    2015-01-01

    In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a ΔagrA deletion mutant were compared in a collection of 10 soil microcosms. The ΔagrA mutant displayed significantly reduced survival in these biotic soil microcosms, and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional, while the variations in the transcriptomes between the wild type and the ΔagrA deletion mutant were modest under abiotic conditions. Indeed, in biotic soil environments, 578 protein-coding genes and an extensive repertoire of noncoding RNAs (ncRNAs) were differentially transcribed. The transcription of genes coding for proteins involved in cell envelope and cellular processes, including the phosphotransferase system and ABC transporters, and proteins involved in resistance to antimicrobial peptides was affected. Under sterilized soil conditions, the differences were limited to 86 genes and 29 ncRNAs. These results suggest that the response regulator AgrA of the Agr communication system plays important roles during the saprophytic life of L. monocytogenes in soil. PMID:26002901

  11. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages.

    PubMed

    Cumley, Nicola J; Smith, Leanne M; Anthony, Mark; May, Robin C

    2012-05-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  12. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  13. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

    PubMed Central

    Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

    1989-01-01

    The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted

  14. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

    PubMed

    Aranda, Juan F; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A

    2011-04-15

    Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes. PMID:21325632

  15. Desnutrin/ATGL is Regulated by AMPK and is Required for a Brown Adipose Phenotype

    PubMed Central

    Ahmadian, Maryam; Abbott, Marcia J.; Tang, Tianyi; Hudak, Carolyn S.S.; Kim, Yangha; Bruss, Matthew; Hellerstein, Marc K.; Lee, Hui-Young; Samuel, Varman T.; Shulman, Gerald I.; Wang, Yuhui; Duncan, Robin E.; Kang, Chulho; Sul, Hei Sook

    2011-01-01

    SUMMARY While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here, we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis. PMID:21641555

  16. PPARα Is Required for PPARδ Action in Regulation of Body Weight and Hepatic Steatosis in Mice

    PubMed Central

    Garbacz, Wojciech G.; Huang, Jeffrey T. J.; Higgins, Larry G.; Wahli, Walter; Palmer, Colin N. A.

    2015-01-01

    Peroxisome proliferator activated receptors alpha (PPARα) and delta (PPARδ) belong to the nuclear receptor superfamily. PPARα is a target of well established lipid-lowering drugs. PPARδ (also known as PPARβ/δ) has been investigated as a promising antidiabetic drug target; however, the evidence in the literature on PPARδ effect on hepatic lipid metabolism is inconsistent. Mice conditionally expressing human PPARδ demonstrated pronounced weight loss and promoted hepatic steatosis when treated with GW501516 (PPARδ-agonist) when compared to wild type mice. This effect was completely absent in mice with either a dominant negative form of PPARδ or deletion of the DNA binding domain of PPARδ. This confirmed the absolute requirement for PPARδ in the physiological actions of GW501516 and confirmed the potential utility against the human form of this receptor. Surprisingly the genetic deletion of PPARα also abrogated the effect of GW501516 in terms of both weight loss and hepatic lipid accumulation. Also the levels of the PPARα endogenous agonist 16:0/18:1-GPC were shown to be modulated by PPARδ in wild type mice. Our results show that both PPARδ and PPARα receptors are essential for GW501516-driven adipose tissue reduction and subsequently hepatic steatosis, with PPARα working downstream of PPARδ. PMID:26604919

  17. Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress.

    PubMed

    McGrail, Daniel J; McAndrews, Kathleen M; Brandenburg, Chandler P; Ravikumar, Nithin; Kieu, Quang Minh N; Dawson, Michelle R

    2015-10-01

    For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress. PMID:26445434

  18. The Extracellular-Regulated Kinase Effector Lk6 is Required for Glutamate Receptor Localization at the Drosophila Neuromuscular Junction.

    PubMed

    Hussein, Nizar A; Delaney, Taylor L; Tounsel, Brittany L; Liebl, Faith L W

    2016-01-01

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). We used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. Our data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. We also find that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. PMID:27199570

  19. Induction of somatic embryos in Arabidopsis requires local YUCCA expression mediated by the down-regulation of ethylene biosynthesis.

    PubMed

    Bai, Bo; Su, Ying Hua; Yuan, Jia; Zhang, Xian Sheng

    2013-07-01

    Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results in somatic embryogenesis, the mechanisms underlying the initiation of somatic embryos (SEs) are poorly understood. In this study, we showed that YUCCAs (YUCs) encoding key enzymes in auxin biosynthesis are required for SE induction in Arabidopsis. To identify other factors mediating SE initiation, we performed transcriptional profiling and gene expression analysis. The results showed that genes involved in ethylene biosynthesis and its responses were down-regulated during SE initiation. Ethylene level decreased progressively during SE initiation, whereas treatment with the metabolic precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), or mutation of ETHYLENE-OVERPRODUCTION1 (ETO1) disrupted SE induction, suggesting that ethylene plays a role in this process. Suppression of SE induction was also observed in the constitutive triple response 1 (ctr1) mutant, in which ethylene signaling was enhanced. These results indicate that down-regulation of not only ethylene biosynthesis, but also ethylene response is critical for SE induction. We further showed that ethylene disturbed SE initiation through inhibiting YUC expression that might be involved in local auxin biosynthesis and subsequent auxin distribution. Our results provide new information on the mechanisms of hormone-regulated SE initiation. PMID:23271028

  20. The Extracellular-Regulated Kinase Effector Lk6 is Required for Glutamate Receptor Localization at the Drosophila Neuromuscular Junction

    PubMed Central

    Hussein, Nizar A.; Delaney, Taylor L.; Tounsel, Brittany L.; Liebl, Faith L.W.

    2016-01-01

    The proper localization and synthesis of postsynaptic glutamate receptors are essential for synaptic plasticity. Synaptic translation initiation is thought to occur via the target of rapamycin (TOR) and mitogen-activated protein kinase signal-integrating kinase (Mnk) signaling pathways, which is downstream of extracellular-regulated kinase (ERK). We used the model glutamatergic synapse, the Drosophila neuromuscular junction, to better understand the roles of the Mnk and TOR signaling pathways in synapse development. These synapses contain non-NMDA receptors that are most similar to AMPA receptors. Our data show that Lk6, the Drosophila homolog of Mnk1 and Mnk2, is required in either presynaptic neurons or postsynaptic muscle for the proper localization of the GluRIIA glutamate receptor subunit. Lk6 may signal through eukaryotic initiation factor (eIF) 4E to regulate the synaptic levels of GluRIIA as either interfering with eIF4E binding to eIF4G or expression of a nonphosphorylatable isoform of eIF4E resulted in a significant reduction in GluRIIA at the synapse. We also find that Lk6 and TOR may independently regulate synaptic levels of GluRIIA. PMID:27199570

  1. Bidirectional regulation of synaptic transmission by BRAG1/IQSEC2 and its requirement in long-term depression

    PubMed Central

    Brown, Joshua C.; Petersen, Amber; Zhong, Ling; Himelright, Miranda L.; Murphy, Jessica A.; Walikonis, Randall S.; Gerges, Nashaat Z.

    2016-01-01

    Dysfunction of the proteins regulating synaptic function can cause synaptic plasticity imbalance that underlies neurological disorders such as intellectual disability. A study found that four distinct mutations within BRAG1, an Arf-GEF synaptic protein, each led to X-chromosome-linked intellectual disability (XLID). Although the physiological functions of BRAG1 are poorly understood, each of these mutations reduces BRAG1's Arf-GEF activity. Here we show that BRAG1 is required for the activity-dependent removal of AMPA receptors in rat hippocampal pyramidal neurons. Moreover, we show that BRAG1 bidirectionally regulates synaptic transmission. On one hand, BRAG1 is required for the maintenance of synaptic transmission. On the other hand, BRAG1 expression enhances synaptic transmission, independently of BRAG1 Arf-GEF activity or neuronal activity, but dependently on its C-terminus interactions. This study demonstrates a dual role of BRAG1 in synaptic function and highlights the functional relevance of reduced BRAG1 Arf-GEF activity as seen in the XLID-associated human mutations. PMID:27009485

  2. The Switch Regulating Transcription of the Escherichia coli Biotin Operon Does Not Require Extensive Protein-Protein Interactions

    PubMed Central

    Solbiati, José; Cronan, John E.

    2009-01-01

    Transcription of the Escherichia coli biotin (bio) operon is regulated by BirA, a protein that is not only the repressor that regulates bio operon expression by DNA binding but also the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein that is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (bio-AMP), the obligatory intermediate of the attachment reaction. The current model postulates that the unmodified acceptor protein binds the monomeric BirA:bio-AMP complex and thereby blocks assembly (dimerization) of the form of BirA that binds DNA. We report that expression of fusion proteins that carry synthetic biotin accepting peptide sequences was as effective as the natural acceptor protein in derepression of bio operon transcription. These peptide sequences have sequences that are remarkably dissimilar to that of the natural acceptor protein and thus our data argue that the regulatory switch does not require the extensive protein-protein interactions postulated in the current model. PMID:20142036

  3. Regulated Expression of miR-155 is Required for iNKT Cell Development

    PubMed Central

    Burocchi, Alessia; Pittoni, Paola; Tili, Esmerina; Rigoni, Alice; Costinean, Stefan; Croce, Carlo Maria; Colombo, Mario Paolo

    2015-01-01

    Invariant natural killer T cells (iNKT cells) are CD1d-restricted, lipid antigen-reactive T lymphocytes with immunoregulatory functions. iNKT cell development in the thymus proceeds through subsequent stages, defined by the expression of CD44 and NK1.1, and is dictated by a unique gene expression program, including microRNAs. Here, we investigated whether miR-155, a microRNA involved in differentiation of most hematopoietic cells, played any role in iNKT cell development. To this end, we assessed the expression of miR-155 along iNKT cell maturation in the thymus, and studied the effects of miR-155 on iNKT cell development using Lck-miR-155 transgenic mice, which over express miR-155 in T cell lineage under the lymphocyte-specific protein tyrosine kinase (Lck) promoter. We show that miR-155 is expressed by newly selected immature wild-type iNKT cells and turned off along iNKT cells differentiation. In transgenic mice, miR-155 over-expression resulted in a substantial block of iNKT cell maturation at Stage 2, in the thymus toward an overall reduction of peripheral iNKT cells, unlike mainstream T cells. Furthermore, the effects of miR-155 over-expression on iNKT cell differentiation were cell autonomous. Finally, we identified Ets1 and ITK transcripts as relevant targets of miR-155 in iNKT cell differentiation. Altogether, these results demonstrate that a tight control of miR-155 expression is required for the development of iNKT cells. PMID:25870598

  4. Cortactin Is Required for N-cadherin Regulation of Kv1.5 Channel Function*

    PubMed Central

    Cheng, Lan; Yung, Aaron; Covarrubias, Manuel; Radice, Glenn L.

    2011-01-01

    The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K+ channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, IK,slow density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice. PMID:21507952

  5. Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

    PubMed Central

    Wold, Mitchell S.; Gong, Shiaoching; Phillips, Greg R.; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

    2014-01-01

    Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis. PMID:25275521

  6. The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa.

    PubMed Central

    Nowrousian, Minou; Duffield, Giles E; Loros, Jennifer J; Dunlap, Jay C

    2003-01-01

    The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature. PMID:12871904

  7. CovR and VicRK regulate cell surface biogenesis genes required for biofilm formation in Streptococcus mutans.

    PubMed

    Stipp, Rafael N; Boisvert, Heike; Smith, Daniel J; Höfling, José F; Duncan, Margaret J; Mattos-Graner, Renata O

    2013-01-01

    The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic) or covR (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA), or regulated only by VicR (SMU.2146c) or CovR (epsC) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose. PMID:23554881

  8. CovR and VicRK Regulate Cell Surface Biogenesis Genes Required for Biofilm Formation in Streptococcus mutans

    PubMed Central

    Stipp, Rafael N.; Boisvert, Heike; Smith, Daniel J.; Höfling, José F.; Duncan, Margaret J.; Mattos-Graner, Renata O.

    2013-01-01

    The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic) or covR (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA), or regulated only by VicR (SMU.2146c) or CovR (epsC) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose. PMID:23554881

  9. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  10. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    DOE PAGESBeta

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

  11. 25 CFR 518.7 - If a tribe holds a certificate of self-regulation, is it required to report information to the...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ....7 Indians NATIONAL INDIAN GAMING COMMISSION, DEPARTMENT OF THE INTERIOR GENERAL PROVISIONS SELF REGULATION OF CLASS II GAMING § 518.7 If a tribe holds a certificate of self-regulation, is it required to... signed by an authorized tribal official, which explains how tribal net gaming revenues were used...

  12. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  13. The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels

    SciTech Connect

    Hwang, Mi-Na; Min, Chan-Hee; Kim, Hyung Sik; Lee, Ho; Yoon, Kyong-Ah; Park, Sung Yong; Lee, Eun Sook; Yoon, Sungpil . E-mail: yoons@ncc.re.kr

    2007-08-24

    We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.

  14. Myxococcus xanthus mokA Encodes a Histidine Kinase-Response Regulator Hybrid Sensor Required for Development and Osmotic Tolerance

    PubMed Central

    Kimura, Yoshio; Nakano, Hiromi; Terasaka, Hideaki; Takegawa, Kaoru

    2001-01-01

    A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain. mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum. PMID:11157925

  15. Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist.

    PubMed Central

    Movahedzadeh, F; Colston, M J; Davis, E O

    1997-01-01

    The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence. PMID:9171394

  16. Binding and Transcriptional Regulation by 14-3-3 (Bmh) Proteins Requires Residues Outside of the Canonical Motif

    PubMed Central

    Parua, Pabitra K.

    2014-01-01

    Evolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations, ADR1c alleles that alter activator-dependent gene expression. Here, we report that ADR1c alleles and other mutations in the regulatory domain impair Bmh binding and abolish Bmh-dependent regulation both directly and indirectly. The indirect effect is caused by mutations that inhibit phosphorylation of Ser230 and thus inhibit Bmh binding, which requires phosphorylated Ser230. However, several mutations inhibit Bmh binding without inhibiting phosphorylation and thus define residues that provide important interaction sites between Adr1 and Bmh. Our proposed model of the Adr1 regulatory domain bound to Bmh suggests that residues Ser238 and Tyr239 could provide cross-dimer contacts to stabilize the complex and that this might explain the failure of a dimerization-deficient Bmh mutant to bind Adr1 and to inhibit its activity. A bioinformatics analysis of Bmh-interacting proteins suggests that residues outside the canonical 14-3-3 motif might be a general property of Bmh target proteins and might help explain the ability of 14-3-3 to distinguish target and nontarget proteins. Bmh binding to the Adr1 regulatory domain, and its failure to bind when mutations are present, explains at a molecular level the transcriptional phenotype of ADR1c mutants. PMID:24142105

  17. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

    PubMed Central

    Bilder, Patrick; Sun, Meihao; Lim, Jihyeon; Bielefeldt-Ohmann, Helle; Basaraba, Randall; So, Melvin; Zhu, Guofeng; Tufariello, JoAnn M.; Izzo, Angelo A.; Orme, Ian M.; Almo, Steve C.; Leyh, Thomas S.; Chan, John

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection. PMID:19478878

  18. Transcriptome Reprogramming by Plasmid-Encoded Transcriptional Regulators Is Required for Host Niche Adaption of a Macrophage Pathogen

    PubMed Central

    Coulson, Garry B.; Miranda-CasoLuengo, Aleksandra A.; Miranda-CasoLuengo, Raúl; Wang, Xiaoguang; Oliver, Jenna; Willingham-Lane, Jennifer M.

    2015-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte. PMID:26015480

  19. Transcriptome reprogramming by plasmid-encoded transcriptional regulators is required for host niche adaption of a macrophage pathogen.

    PubMed

    Coulson, Garry B; Miranda-CasoLuengo, Aleksandra A; Miranda-CasoLuengo, Raúl; Wang, Xiaoguang; Oliver, Jenna; Willingham-Lane, Jennifer M; Meijer, Wim G; Hondalus, Mary K

    2015-08-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte. PMID:26015480

  20. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    SciTech Connect

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  1. Epigenetic regulation of Atrophin1 by lysine-specific demethylase 1 is required for cortical progenitor maintenance

    PubMed Central

    Zhang, Feng; Xu, Dan; Yuan, Ling; Sun, Yiming; Xu, Zhiheng

    2014-01-01

    Lysine-specific demethylase 1 (LSD1) is involved in gene regulation and development; however, its precise function, molecular targets and underlying mechanisms during development are poorly understood. Here we show that LSD1 is required for neuronal progenitor cell (NPC) maintenance during cortical development. A ChIP-seq analysis identified a LSD1-binding site (LBAL) downstream of Atrophin1 (ATN1). Surprisingly, tranylcypromine (LSD1 inhibitor) treatment increased H3K4 methylation at LBAL, leading to ATN1 repression and NPC differentiation. Knockdown of LSD1 and ATN1 phenocopied each other in inducing NPC premature differentiation and depletion, which could be rescued by ATN1 overexpression, suggesting that LSD1 controls NPC differentiation via regulation of ATN1 methylation status and expression. The involvement of LSD1 in ATN1 expression and NPC maintenance were confirmed in knockout mice. These findings hint at the potential application for the clinical drug, tranylcypromine, in the prevention and/or treatment of ATN1-associated degenerative disease, dentatorubral-pallidoluysian atrophy. PMID:25519973

  2. Interleukin33 Is Required for Disposal of Unnecessary Cells during Ovarian Atresia through Regulation of Autophagy and Macrophage Migration

    PubMed Central

    Wu, Jean; Carlock, Colin; Zhou, Cindy; Nakae, Susumu; Hicks, John; Adams, Henry P.; Lou, Yahuan

    2015-01-01

    Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. Interleukin33 (IL33) is a member of the IL1 cytokine gene family. Consecutive expression of IL33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL33 expression patterns and ovarian atresia. Here, we demonstrated that IL33 is required for disposal of degenerative tissue during ovarian atresia using Il33−/− mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33−/− mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive lifespan was shortened to 2/3 of that for Il33+/− littermates. IL33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provided direct evidence supporting an expanded role of IL33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells. PMID:25617473

  3. The TORMOZ Gene Encodes a Nucleolar Protein Required for Regulated Division Planes and Embryo Development in Arabidopsis[W

    PubMed Central

    Griffith, Megan E.; Mayer, Ulrike; Capron, Arnaud; Ngo, Quy A.; Surendrarao, Anandkumar; McClinton, Regina; Jürgens, Gerd; Sundaresan, Venkatesan

    2007-01-01

    Embryogenesis in Arabidopsis thaliana is marked by a predictable sequence of oriented cell divisions, which precede cell fate determination. We show that mutation of the TORMOZ (TOZ) gene yields embryos with aberrant cell division planes and arrested embryos that appear not to have established normal patterning. The defects in toz mutants differ from previously described mutations that affect embryonic cell division patterns. Longitudinal division planes of the proembryo are frequently replaced by transverse divisions and less frequently by oblique divisions, while divisions of the suspensor cells, which divide only transversely, appear generally unaffected. Expression patterns of selected embryo patterning genes are altered in the mutant embryos, implying that the positional cues required for their proper expression are perturbed by the misoriented divisions. The TOZ gene encodes a nucleolar protein containing WD repeats. Putative TOZ orthologs exist in other eukaryotes including Saccharomyces cerevisiae, where the protein is predicted to function in 18S rRNA biogenesis. We find that disruption of the Sp TOZ gene results in cell division defects in Schizosaccharomyces pombe. Previous studies in yeast and animal cells have identified nucleolar proteins that regulate the exit from M phase and cytokinesis, including factors involved in pre-rRNA processing. Our study suggests that in plant cells, nucleolar functions might interact with the processes of regulated cell divisions and influence the selection of longitudinal division planes during embryogenesis. PMID:17616738

  4. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly

    PubMed Central

    Racine, Pierre-Jean; Chamontin, Célia; de Rocquigny, Hugues; Bernacchi, Serena; Paillart, Jean-Christophe; Mougel, Marylène

    2016-01-01

    HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT. PMID:27273064

  5. FgFlbD regulates hyphal differentiation required for sexual and asexual reproduction in the ascomycete fungus Fusarium graminearum.

    PubMed

    Son, Hokyoung; Kim, Myung-Gu; Chae, Suhn-Kee; Lee, Yin-Won

    2014-11-01

    Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi. PMID:25277408

  6. HBx induces hypomethylation of distal intragenic CpG islands required for active expression of developmental regulators.

    PubMed

    Lee, Sun-Min; Lee, Young-gun; Bae, Jae-Bum; Choi, Jung Kyoon; Tayama, Chiharu; Hata, Kenichiro; Yun, Yungdae; Seong, Je-Kyung; Kim, Young-Joon

    2014-07-01

    Epigenetic alterations caused by viral oncoproteins are strong initiation factors for cancer development, but their mechanisms are largely unknown. To identify the epigenetic effects of viral hepatitis B virus X (HBx) that lead to hepatocellular carcinoma (HCC), we profiled the DNA methylomes of normal and HBx transgenic mouse liver. Intriguingly, severe hypomethylation of intragenic CpG islands (CGIs) was observed in HBx liver before the full development of HCC. Normally, these CGIs were highly methylated (mCGIs) by the DNMT3L complex and marked with epigenetic signatures associated with active expression, such as H3K36me3. Hypomethylation of mCGI was caused by the downregulation of Dnmt3L and Dnmt3a due to HBx bound to their promoters, along with HDAC1. These events lead to the downregulation of many developmental regulators that could facilitate tumorigenesis. Here we provide an intriguing epigenetic regulation mediated by mCGI that is required for cell differentiation and describe a previously unidentified epigenetic role for HBx in promoting HCC development. PMID:24941955

  7. Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

    PubMed

    Racine, Pierre-Jean; Chamontin, Célia; de Rocquigny, Hugues; Bernacchi, Serena; Paillart, Jean-Christophe; Mougel, Marylène

    2016-01-01

    HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT. PMID:27273064

  8. A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

    PubMed

    Tian, Xuejun; Gala, Upasana; Zhang, Yongping; Shang, Weina; Nagarkar Jaiswal, Sonal; di Ronza, Alberto; Jaiswal, Manish; Yamamoto, Shinya; Sandoval, Hector; Duraine, Lita; Sardiello, Marco; Sillitoe, Roy V; Venkatachalam, Kartik; Fan, Hengyu; Bellen, Hugo J; Tong, Chao

    2015-03-01

    Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis. PMID:25811491

  9. A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

    PubMed Central

    Zhang, Yongping; Shang, Weina; Nagarkar Jaiswal, Sonal; di Ronza, Alberto; Jaiswal, Manish; Yamamoto, Shinya; Sandoval, Hector; Duraine, Lita; Sardiello, Marco; Sillitoe, Roy V.; Venkatachalam, Kartik; Fan, Hengyu; Bellen, Hugo J.; Tong, Chao

    2015-01-01

    Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis. PMID:25811491

  10. Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells.

    PubMed

    Gidda, Satinder K; Park, Sunjung; Pyc, Michal; Yurchenko, Olga; Cai, Yingqi; Wu, Peng; Andrews, David W; Chapman, Kent D; Dyer, John M; Mullen, Robert T

    2016-04-01

    Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth. PMID:26896396

  11. Greater-than-Class C low-level radioactive waste transportation regulations and requirements study. National Low-Level Waste Management Program

    SciTech Connect

    Tyacke, M.; Schmitt, R.

    1993-07-01

    The purpose of this report is to identify the regulations and requirements for transporting greater-than-Class C (GTCC) low-level radioactive waste (LLW) and to identify planning activities that need to be accomplished in preparation for transporting GTCC LLW. The regulations and requirements for transporting hazardous materials, of which GTCC LLW is included, are complex and include several Federal agencies, state and local governments, and Indian tribes. This report is divided into five sections and three appendices. Section 1 introduces the report. Section 2 identifies and discusses the transportation regulations and requirements. The regulations and requirements are divided into Federal, state, local government, and Indian tribes subsections. This report does not identify the regulations or requirements of specific state, local government, and Indian tribes, since the storage, treatment, and disposal facility locations and transportation routes have not been specifically identified. Section 3 identifies the planning needed to ensure that all transportation activities are in compliance with the regulations and requirements. It is divided into (a) transportation packaging; (b) transportation operations; (c) system safety and risk analysis, (d) route selection; (e) emergency preparedness and response; and (f) safeguards and security. This section does not provide actual planning since the details of the Department of Energy (DOE) GTCC LLW Program have not been finalized, e.g., waste characterization and quantity, storage, treatment and disposal facility locations, and acceptance criteria. Sections 4 and 5 provide conclusions and referenced documents, respectively.

  12. Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter.

    PubMed Central

    MacMorris, M; Broverman, S; Greenspoon, S; Lea, K; Madej, C; Blumenthal, T; Spieth, J

    1992-01-01

    The Caenorhabditis elegans vitellogenin genes are subject to sex-, stage-, and tissue-specific regulation: they are expressed solely in the adult hermaphrodite intestine. Comparative sequence analysis of the DNA immediately upstream of these genes revealed the presence of two repeated heptameric elements, vit promoter element 1 (VPE1) and VPE2. VPE1 has the consensus sequence TGTCAAT, while VPE2, CTGATAA, shares the recognition sequence of the GATA family of transcription factors. We report here a functional analysis of the VPEs within the 5'-flanking region of the vit-2 gene using stable transgenic lines. The 247 upstream bp containing the VPEs was sufficient for high-level, regulated expression. Furthermore, none of the four deletion mutations or eight point mutations tested resulted in expression of the reporter gene in larvae, males, or inappropriate hermaphrodite tissues. Mutation of the VPE1 closest to the TATA box inactivated the promoter, in spite of the fact that four additional close matches to the VPE1 consensus sequence are present within the 5'-flanking 200 bp. Each of these upstream VPE1-like sequences could be mutated without loss of high-level transgene expression, suggesting that if these VPE1 sequences play a role in regulating vit-2, their effects are more subtle. A site-directed mutation in the overlapping VPE1 and VPE2 at -98 was sufficient to inactivate the promoter, indicating that one or both of these VPEs must be present for activation of vit-2 transcription. Similarly, a small perturbation of the VPE2 at -150 resulted in reduction of fp155 expression, while a more extensive mutation in this element eliminated expression. On the other hand, deletion of this VPE2 and all upstream DNA still permitted correctly regulated expression, although at a very low level, suggesting that this VPE2 performs an important role in activation of vit-2 expression but may not be absolutely required. The results, taken together, demonstrate that both VPE1 and

  13. Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter.

    PubMed

    MacMorris, M; Broverman, S; Greenspoon, S; Lea, K; Madej, C; Blumenthal, T; Spieth, J

    1992-04-01

    The Caenorhabditis elegans vitellogenin genes are subject to sex-, stage-, and tissue-specific regulation: they are expressed solely in the adult hermaphrodite intestine. Comparative sequence analysis of the DNA immediately upstream of these genes revealed the presence of two repeated heptameric elements, vit promoter element 1 (VPE1) and VPE2. VPE1 has the consensus sequence TGTCAAT, while VPE2, CTGATAA, shares the recognition sequence of the GATA family of transcription factors. We report here a functional analysis of the VPEs within the 5'-flanking region of the vit-2 gene using stable transgenic lines. The 247 upstream bp containing the VPEs was sufficient for high-level, regulated expression. Furthermore, none of the four deletion mutations or eight point mutations tested resulted in expression of the reporter gene in larvae, males, or inappropriate hermaphrodite tissues. Mutation of the VPE1 closest to the TATA box inactivated the promoter, in spite of the fact that four additional close matches to the VPE1 consensus sequence are present within the 5'-flanking 200 bp. Each of these upstream VPE1-like sequences could be mutated without loss of high-level transgene expression, suggesting that if these VPE1 sequences play a role in regulating vit-2, their effects are more subtle. A site-directed mutation in the overlapping VPE1 and VPE2 at -98 was sufficient to inactivate the promoter, indicating that one or both of these VPEs must be present for activation of vit-2 transcription. Similarly, a small perturbation of the VPE2 at -150 resulted in reduction of fp155 expression, while a more extensive mutation in this element eliminated expression. On the other hand, deletion of this VPE2 and all upstream DNA still permitted correctly regulated expression, although at a very low level, suggesting that this VPE2 performs an important role in activation of vit-2 expression but may not be absolutely required. The results, taken together, demonstrate that both VPE1 and

  14. Regulation of cellular diacylglycerol through lipid phosphate phosphatases is required for pathogenesis of the rice blast fungus, Magnaporthe oryzae.

    PubMed

    Sadat, Md Abu; Jeon, Junhyun; Mir, Albely Afifa; Choi, Jaeyoung; Choi, Jaehyuk; Lee, Yong-Hwan

    2014-01-01

    Considering implication of diacylglycerol in both metabolism and signaling pathways, maintaining proper levels of diacylglycerol (DAG) is critical to cellular homeostasis and development. Except the PIP2-PLC mediated pathway, metabolic pathways leading to generation of DAG converge on dephosphorylation of phosphatidic acid catalyzed by lipid phosphate phosphatases. Here we report the role of such enzymes in a model plant pathogenic fungus, Magnaporthe oryzae. We identified five genes encoding putative lipid phosphate phosphatases (MoLPP1 to MoLPP5). Targeted disruption of four genes (except MoLPP4) showed that MoLPP3 and MoLPP5 are required for normal progression of infection-specific development and proliferation within host plants, whereas MoLPP1 and MoLPP2 are indispensable for fungal pathogenicity. Reintroduction of MoLPP3 and MoLPP5 into individual deletion mutants restored all the defects. Furthermore, exogenous addition of saturated DAG not only restored defect in appressorium formation but also complemented reduced virulence in both mutants. Taken together, our data indicate differential roles of lipid phosphate phosphatase genes and requirement of proper regulation of cellular DAGs for fungal development and pathogenesis. PMID:24959955

  15. Development of guidance on applications of regulatory requirements for regulating large, contaminated equipment and large decommissioning and decontamination (D and D) components

    SciTech Connect

    Pope, R.B.; Easton, E.P.; Cook, J.R.; Boyle, R.W.

    1997-10-01

    In 1985, the International Atomic Energy Agency issued revised regulations for the safe transport of radioactive material. Significant were major changes to requirements for Low Specific Activity material and Surface Contaminated Objects. As these requirements were adopted into regulations in the US, it was recognized that guidance on how to apply these requirements to large, contaminated/activated pieces of equipment and decommissioning and decontamination objects would be needed both by the regulators and those regulated to clarify technical uncertainties and ensure implementation. Thus, the US Department of Transportation and the US Nuclear Regulatory Commission, with assistance of staff from Oak Ridge National Laboratory, are preparing regulatory guidance which will present examples of acceptable methods for demonstrating compliance with the revised rules for large items. Concepts being investigated for inclusion in the pending guidance are discussed in this paper. Under current plans, the guidance will be issued for public comment before final issuance in 1997.

  16. Responses to iron limitation are impacted by light quality and regulated by RcaE in the chromatically acclimating cyanobacterium Fremyella diplosiphon.

    PubMed

    Pattanaik, Bagmi; Busch, Andrea W U; Hu, Pingsha; Chen, Jin; Montgomery, Beronda L

    2014-05-01

    Photosynthetic organisms adapt to environmental fluctuations of light and nutrient availability. Iron is critical for photosynthetic organismal growth, as many cellular processes depend upon iron cofactors. Whereas low iron levels can have deleterious effects, excess iron can lead to damage, as iron is a reactive metal that can result in the production of damaging radicals. Therefore, organisms regulate cellular iron levels to maintain optimal iron homeostasis. In particular, iron is an essential factor for the function of photosystems associated with photosynthetic light-harvesting complexes. Photosynthetic organisms, including cyanobacteria, generally respond to iron deficiency by reduced growth, degradation of non-essential proteins and in some cases alterations of cellular morphology. In response to fluctuations in ambient light quality, the cyanobacterium Fremyella diplosiphon undergoes complementary chromatic adaptation (CCA). During CCA, phycobiliprotein composition of light-harvesting antennae is altered in response to green light (GL) and red light (RL) for efficient utilization of light energy for photosynthesis. We observed light-regulated responses to iron limitation in F. diplosiphon. RL-grown cells exhibited significant reductions in growth and pigment levels, and alterations in iron-associated proteins, which impact the accumulation of reactive oxygen species under iron-limiting conditions, whereas GL-grown cells exhibited partial resistance to iron limitation. We investigated the roles of known CCA regulators RcaE, RcaF and RcaC in this light-dependent iron-acclimation response. Through comparative analyses of wild-type and CCA mutant strains, we determined that photoreceptor RcaE has a central role in light-induced oxidative stress associated with iron limitation, and impacts light-regulated iron-acclimation responses, physiologically and morphologically. PMID:24623652

  17. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection

    PubMed Central

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection. PMID:26670613

  18. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection.

    PubMed

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection. PMID:26670613

  19. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

    PubMed Central

    Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

    2016-01-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  20. Distinct p21 requirements for regulating normal and self-reactive T cells through IFN-γ production

    PubMed Central

    Daszkiewicz, Lidia; Vázquez-Mateo, Cristina; Rackov, Gorjana; Ballesteros-Tato, André; Weber, Kathrin; Madrigal-Avilés, Adrián; Di Pilato, Mauro; Fotedar, Arun; Fotedar, Rati; Flores, Juana M.; Esteban, Mariano; Martínez-A, Carlos; Balomenos, Dimitrios

    2015-01-01

    Self/non-self discrimination characterizes immunity and allows responses against pathogens but not self-antigens. Understanding the principles that govern this process is essential for designing autoimmunity treatments. p21 is thought to attenuate autoreactivity by limiting T cell expansion. Here, we provide direct evidence for a p21 role in controlling autoimmune T cell autoreactivity without affecting normal T cell responses. We studied C57BL/6, C57BL/6/lpr and MRL/lpr mice overexpressing p21 in T cells, and showed reduced autoreactivity and lymphadenopathy in C57BL/6/lpr, and reduced mortality in MRL/lpr mice. p21 inhibited effector/memory CD4+ CD8+ and CD4−CD8− lpr T cell accumulation without altering defective lpr apoptosis. This was mediated by a previously non-described p21 function in limiting T cell overactivation and overproduction of IFN-γ, a key lupus cytokine. p21 did not affect normal T cell responses, revealing differential p21 requirements for autoreactive and normal T cell activity regulation. The underlying concept of these findings suggests potential treatments for lupus and autoimmune lymphoproliferative syndrome, without compromising normal immunity. PMID:25573673

  1. ATP-dependent RecG Helicase Is Required for the Transcriptional Regulator OxyR Function in Pseudomonas species*

    PubMed Central

    Yeom, Jinki; Lee, Yunho; Park, Woojun

    2012-01-01

    The oxyR gene appears to reside in an operon with the recG helicase gene in many bacteria, including pathogenic Pseudomonas aeruginosa and Pseudomonas putida. Analysis of P. putida transcriptomes shows that many OxyR-controlled genes are regulated by the ATP-dependent RecG helicase and that RecG alone modulates the expression of many genes. We found that purified RecG binds to the promoters of many OxyR-controlled genes and that expression of these genes was not induced under conditions of oxidative stress in recG mutants of P. aeruginosa, P. putida, and Escherichia coli. In vitro data revealed that promoters containing palindromic sequences are essential for RecG binding and that single-strand binding proteins and ATP are also needed for RecG to promote transcription, whereas a magnesium ion has the opposite effect. The OxyR tetramer preferentially binds to promoters after RecG has generated linear DNA in the presence of ATP; otherwise, the OxyR dimer has higher affinity. This study provides new insights into the mechanism of bacterial transcription by demonstrating that RecG might be required for the induction of the OxyR regulon by unwinding palindromic DNA for transcription. This work describes a novel bacterial transcriptional function by RecG helicase with OxyR and may provide new targets for controlling Pseudomonas species pathogen. PMID:22621928

  2. HIF- and Non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila melanogaster

    PubMed Central

    Li, Yan; Padmanabha, Divya; Gentile, Luciana B.; Dumur, Catherine I.; Beckstead, Robert B.; Baker, Keith D.

    2013-01-01

    Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs. PMID:23382692

  3. Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis.

    PubMed

    Mancias, Joseph D; Pontano Vaites, Laura; Nissim, Sahar; Biancur, Douglas E; Kim, Andrew J; Wang, Xiaoxu; Liu, Yu; Goessling, Wolfram; Kimmelman, Alec C; Harper, J Wade

    2015-01-01

    NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. PMID:26436293

  4. Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis

    PubMed Central

    Mancias, Joseph D; Pontano Vaites, Laura; Nissim, Sahar; Biancur, Douglas E; Kim, Andrew J; Wang, Xiaoxu; Liu, Yu; Goessling, Wolfram; Kimmelman, Alec C; Harper, J Wade

    2015-01-01

    NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting. DOI: http://dx.doi.org/10.7554/eLife.10308.001 PMID:26436293

  5. Zinc fingers 1, 2, 5 and 6 of transcriptional regulator, PRDM4, are required for its nuclear localisation.

    PubMed

    Tunbak, Hale; Georgiou, Christiana; Guan, Cui; Richardson, William David; Chittka, Alexandra

    2016-05-27

    PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclear localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5-6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. PMID:27125459

  6. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis

    PubMed Central

    Jeanblanc, Jerome; Logrip, Marian L.; Janak, Patricia H.; Ron, Dorit

    2013-01-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-γ or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  7. Down-regulation of NR3A-containing NMDARs is required for synapse maturation and memory consolidation

    PubMed Central

    Roberts, Adam C.; Díez-García, Javier; Rodriguiz, Ramona M.; López, Iciar Paula; Luján, Rafael; Martínez-Turrillas, Rebeca; Picó, Esther; Henson, Maile A.; Bernardo, Danilo R.; Jarrett, Thomas M.; Clendeninn, Dallis J.; López-Mascaraque, Laura; Feng, Guoping; Lo, Donald C.; Wesseling, John F.; Wetsel, William C.; Philpot, Benjamin D.; Pérez-Otaño, Isabel

    2012-01-01

    SUMMARY NR3A is the only NMDA receptor (NMDAR) subunit that down-regulates sharply prior to the onset of sensitive periods for plasticity, yet the functional importance of this transient expression remains largely unknown. To investigate the possibility that removal/replacement of juvenile NR3A-containing NMDARs is involved in experience-driven synapse maturation, we used a reversible transgenic system that allowed persistent NR3A expression in the postnatal forebrain. We found that removal of NR3A is required to develop strong NMDAR currents, full expression of long-term synaptic plasticity, a mature synaptic organization characterized by more synapses and larger postsynaptic densities, and the ability to form long-term memories. Deficits associated with prolonged NR3A were reversible, as late-onset suppression of transgene expression rescued both the synaptic and memory impairments. Our results suggest that NR3A behaves as a molecular brake to prevent the premature strengthening and stabilization of excitatory synapses, and that NR3A removal might thereby initiate critical stages of synapse maturation during early postnatal neural development. PMID:19679074

  8. RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

    PubMed

    Peterson, Celeste N; Ruiz, Natividad; Silhavy, Thomas J

    2004-11-01

    In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate. PMID:15489452

  9. Guide to NRC reporting and recordkeeping requirements. Compiled from requirements in Title 10 of the U.S. Code of Federal Regulations as codified on December 31, 1993; Revision 1

    SciTech Connect

    Collins, M.; Shelton, B.

    1994-07-01

    This compilation includes in the first two sections the reporting and recordkeeping requirements applicable to US Nuclear Regulatory Commission (NRC) licensees and applicants and to members of the public. It includes those requirements codified in Title 10 of the code of Federal Regulations, Chapter 1, on December 31, 1993. It also includes, in a separate section, any of those requirements that were superseded or discontinued between January 1992 and December 1993. Finally, the appendix lists mailing and delivery addresses for NRC Headquarters and Regional Offices mentioned in the compilation. The Office of Information Resources Management staff compiled this listing of reporting and recordkeeping requirements to briefly describe each in a single document primarily to help licensees readily identify the requirements. The compilation is not a substitute for the regulations, and is not intended to impose any new requirements or technical positions. It is part of NRC`s continuing efforts to comply with the Paperwork Reduction Act of 1980 and the Office of Management and Budget regulations that mandate effective and efficient Federal information resources management programs.

  10. Eukaryotic Translation Initiation Factor eIFiso4G Is Required to Regulate Violaxanthin De-epoxidase Expression in Arabidopsis*

    PubMed Central

    Chen, Zhong; Jolley, Blair; Caldwell, Christian; Gallie, Daniel R.

    2014-01-01

    The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity. PMID:24706761

  11. Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

    PubMed

    Miyoshi, Yuka; Tanabe, Soichi; Suzuki, Takuya

    2016-07-01

    Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription. PMID:27151944

  12. Type-A response regulators are required for proper root apical meristem function through post-transcriptional regulation of PIN auxin efflux carriers.

    PubMed

    Zhang, Wenjing; To, Jennifer P C; Cheng, Chia-Yi; Schaller, G Eric; Kieber, Joseph J

    2011-10-01

    The phytohormones cytokinin and auxin regulate a diverse array of plant processes, often acting together to modulate growth and development. Although much has been learned with regard to how each of these hormones act individually, we are just beginning to understand how these signals interact to achieve an integrated response. Previous studies indicated that exogenous cytokinin has an effect on the transcription of several PIN efflux carriers. Here we show that disruption of type-A Arabidopsis response regulators (ARRs), which are negative regulators of cytokinin signalling, alters the levels of PIN proteins and results in increased sensitivity to N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport. Disruption of eight of the 10 type-A ARR genes affects root development by altering the size of the apical meristem. Furthermore, we show that the effect of cytokinin on PIN abundance occurs primarily at the post-transcriptional level. Alterations of PIN levels in the type-A ARR mutants result in changes in the distribution of auxin in root tips as measured by a DR5::GFP reporter, and an altered pattern of cell division and differentiation in the stem cell niche in the root apical meristem. Together, these data indicate that cytokinin, acting through the type-A ARRs, alters the level of several PIN efflux carriers, and thus regulates the distribution of auxin within the root tip. PMID:21645147

  13. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

  14. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... year); (iv) Economic losses associated with pests of concern in the country; (v) Pest biology...

  15. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1, that regulates markedly different genes in different fungi. Despite these differences, many of the regulated genes encode effector proteins or proteins involved in the synthesis of secondary m...

  16. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... with crop production, entomology, plant pathology, and other relevant characteristics of the...

  17. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... with crop production, entomology, plant pathology, and other relevant characteristics of the...

  18. 7 CFR 319.5 - Requirements for submitting requests to change the regulations in 7 CFR part 319.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... regulations in 7 CFR part 319. 319.5 Section 319.5 Agriculture Regulations of the Department of Agriculture... 7 CFR part 319. (a) Definitions. Commodity. A plant, plant product, or other agricultural product... with crop production, entomology, plant pathology, and other relevant characteristics of the...

  19. 41 CFR 101-1.4902-2053 - GSA Form 2053, Agency Consolidated Requirements for GSA Regulations and Other External Issuances.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 41 Public Contracts and Property Management 2 2011-07-01 2007-07-01 true GSA Form 2053, Agency...-2053 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS GENERAL 1-INTRODUCTION 1.49-Illustrations of Forms § 101-1.4902-2053...

  20. 41 CFR 101-1.4902-2053 - GSA Form 2053, Agency Consolidated Requirements for GSA Regulations and Other External Issuances.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 41 Public Contracts and Property Management 2 2014-07-01 2012-07-01 true GSA Form 2053, Agency...-2053 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS GENERAL 1-INTRODUCTION 1.49-Illustrations of Forms § 101-1.4902-2053...

  1. 41 CFR 101-1.4902-2053 - GSA Form 2053, Agency Consolidated Requirements for GSA Regulations and Other External Issuances.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 2 2010-07-01 2010-07-01 true GSA Form 2053, Agency...-2053 Public Contracts and Property Management Federal Property Management Regulations System FEDERAL PROPERTY MANAGEMENT REGULATIONS GENERAL 1-INTRODUCTION 1.49-Illustrations of Forms § 101-1.4902-2053...

  2. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1 in Fusarium oxysporum, that regulates markedly different gene sets in different fungi. Despite these differences, many of the regulated genes affect pathogenicity as they encode effector protei...

  3. 40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 19 2011-07-01 2011-07-01 false State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations XIII Appendix XIII to Part 86 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM NEW AND IN-USE HIGHWAY VEHICLES AND ENGINES...

  4. 40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 19 2010-07-01 2010-07-01 false State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations XIII Appendix XIII to Part 86 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM NEW AND IN-USE HIGHWAY VEHICLES AND ENGINES...

  5. 40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 20 2013-07-01 2013-07-01 false State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations XIII Appendix XIII to Part 86 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM NEW AND IN-USE HIGHWAY VEHICLES AND ENGINES...

  6. 40 CFR Appendix Xiii to Part 86 - State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 20 2012-07-01 2012-07-01 false State Requirements Incorporated by Reference in Part 86 of the Code of Federal Regulations XIII Appendix XIII to Part 86 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM NEW AND IN-USE HIGHWAY VEHICLES AND ENGINES...

  7. 40 CFR Appendix A to Part 282 - State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 28 2013-07-01 2013-07-01 false State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations A Appendix A to Part 282 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED UNDERGROUND STORAGE TANK PROGRAMS Pt. 282, App. A Appendix A...

  8. 40 CFR Appendix A to Part 282 - State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 27 2014-07-01 2014-07-01 false State Requirements Incorporated by Reference in Part 282 of the Code of Federal Regulations A Appendix A to Part 282 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED UNDERGROUND STORAGE TANK PROGRAMS Pt. 282, App. A Appendix A...

  9. Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1.

    PubMed

    Gade, Padmaja; Manjegowda, Srikanta B; Nallar, Shreeram C; Maachani, Uday B; Cross, Alan S; Kalvakolanu, Dhananjaya V

    2014-11-01

    The death-associated protein kinase 1 (DAPK1) is an important regulator of cell death and autophagy. Recently, we have identified that ATF6, an endoplasmic reticulum-resident transcription factor, in association with the transcription factor CEBP-β, regulates the gamma interferon (IFN-γ)-induced expression of Dapk1 (P. Gade et al., Proc. Natl. Acad. Sci. U. S. A. 109:10316-10321, 2012, doi.org/10.1073/pnas.1119273109). IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β were essential for the formation of a novel transcriptional complex that regulates DAPK1. Here, we report that IFN-γ activates the ASK1-MKK3/MKK6-p38 mitogen-activated protein kinase (MAPK) pathway for controlling the activity of ATF6. The terminal enzyme in this pathway, p38 MAPK, phosphorylates a critical threonine residue in ATF6 upstream of its DNA binding domain. ATF6 mutants defective for p38 MAPK phosphorylation fail to undergo proteolytic processing in the Golgi apparatus and drive IFN-γ-induced gene expression and autophagy. We also show that mice lacking Ask1 are highly susceptible to lethal bacterial infection owing to defective autophagy. Together, these results identify a novel host defense pathway controlled by IFN-γ signaling. PMID:25135476

  10. 77 FR 53870 - Availability of a Legal Entity Identifier Meeting the Requirements of the Regulations of the...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-04

    ... charge, all CICI data and all CICI intellectual property rights. B. Requirements for Designation as the... associated reference data and all CICI intellectual property rights. d. This designation is made for a... Requirements, 77 FR 2136 (Jan. 13, 2012),...

  11. Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule.

    PubMed

    Kreikemeyer, B; Boyle, M D; Buttaro, B A; Heinemann, M; Podbielski, A

    2001-01-01

    A novel growth phase-associated two-component-type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum-sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase-associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr-null mutations in S. aureus, with prolonged expression of extracellular matrix protein-binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild-type fasBCA regulation pattern. This strongly suggested that fasX, a putative non-translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild-type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum-sensing regulation

  12. Fibroblast Growth Factor (FGF) Signaling during Gastrulation Negatively Modulates the Abundance of MicroRNAs That Regulate Proteins Required for Cell Migration and Embryo Patterning*

    PubMed Central

    Bobbs, Alexander S.; Saarela, Aleksi V.; Yatskievych, Tatiana A.; Antin, Parker B.

    2012-01-01

    FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways. PMID:22995917

  13. On the role of subspace zeros in retrospective cost adaptive control of non-square plants

    NASA Astrophysics Data System (ADS)

    Dogan Sumer, E.; Bernstein, Dennis S.

    2015-02-01

    We consider adaptive control of non-square plants, that is, plants that have an unequal number of inputs and outputs. In particular, we focus on retrospective cost adaptive control (RCAC), which is a direct, discrete-time adaptive control algorithm that is applicable to stabilisation, command following, disturbance rejection, and model reference control problems. Previous studies on RCAC have focused on control of square plants. In the square case, RCAC requires knowledge of the first non-zero Markov parameter and the non-minimum-phase (NMP) transmission zeros of the plant, if any. No additional information about the plant or the exogenous signals need be known. The goal of the present paper is to consider RCAC for non-square plants. Unlike the square case, we show that the assumption that the non-square plant is minimum phase does not guarantee closed-loop stability and signal boundedness. The main purpose of this paper is to establish the existence of time-invariant input and output subspaces corresponding to the adaptive controller. In particular, we show that RCAC implicitly squares down non-square plants through pre-/post-compensation of the non-square plant with a constant matrix. We show that, for wide plants, the control input generated by RCAC lies in a time-invariant 'input subspace', which is equivalent to pre-compensating the plant with a constant matrix. On the other hand, for tall plants, we show that the controller update is driven by the output of the plant post-compensated with a constant matrix. Accordingly, in either case, signal boundedness properties of the closed-loop system are determined by the transmission zeros of the squared system, which we call the 'subspace zeros'. To deal with NMP subspace zeros, we introduce a robustness modification, which prevents RCAC from cancelling the NMP subspace zeros.

  14. The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform

    PubMed Central

    Dichmann, Darwin S; Walentek, Peter; Harland, Richard M

    2014-01-01

    SUMMARY Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA-seq, we identify 142 splice changes, mostly intron retention and exon skipping, of which 89% are not in current annotations. A previously not described isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism. PMID:25620705

  15. Fragile X Protein is required for inhibition of insulin signaling and regulates glial-dependent neuroblast reactivation in the developing brain.

    PubMed

    Callan, Matthew A; Clements, Nicole; Ahrendt, Nicholas; Zarnescu, Daniela C

    2012-06-26

    Fragile X syndrome (FXS) is the most common form of inherited mental disability and known cause of autism. It is caused by loss of function for the RNA binding protein FMRP, which has been demonstrated to regulate several aspects of RNA metabolism including transport, stability and translation at synapses. Recently, FMRP has been implicated in neural stem cell proliferation and differentiation both in cultured neurospheres as well as in vivo mouse and fly models of FXS. We have previously shown that FMRP deficient Drosophila neuroblasts upregulate Cyclin E, prematurely exit quiescence, and overproliferate to generate on average 16% more neurons. Here we further investigate FMRP's role during early development using the Drosophila larval brain as a model. Using tissue specific RNAi we find that FMRP is required sequentially, first in neuroblasts and then in glia, to regulate exit from quiescence as measured by Cyclin E expression in the brain. Furthermore, we tested the hypothesis that FMRP controls brain development by regulating the insulin signaling pathway, which has been recently shown to regulate neuroblast exit from quiescence. Our data indicate that phosphoAkt, a readout of insulin signaling, is upregulated in dFmr1 brains at the time when FMRP is required in glia for neuroblast reactivation. In addition, dFmr1 interacts genetically with dFoxO, a transcriptional regulator of insulin signaling. Our results provide the first evidence that FMRP is required in vivo, in glia for neuroblast reactivation and suggest that it may do so by regulating the output of the insulin signaling pathway. This article is part of a Special Issue entitled: RNA-Binding Proteins. PMID:22513101

  16. Requirements for Defining Utility Drive Cycles: An Exploratory Analysis of Grid Frequency Regulation Data for Establishing Battery Performance Testing Standards

    SciTech Connect

    Hafen, Ryan P.; Vishwanathan, Vilanyur V.; Subbarao, Krishnappa; Kintner-Meyer, Michael CW

    2011-10-19

    Battery testing procedures are important for understanding battery performance, including degradation over the life of the battery. Standards are important to provide clear rules and uniformity to an industry. The work described in this report addresses the need for standard battery testing procedures that reflect real-world applications of energy storage systems to provide regulation services to grid operators. This work was motivated by the need to develop Vehicle-to-Grid (V2G) testing procedures, or V2G drive cycles. Likewise, the stationary energy storage community is equally interested in standardized testing protocols that reflect real-world grid applications for providing regulation services. As the first of several steps toward standardizing battery testing cycles, this work focused on a statistical analysis of frequency regulation signals from the Pennsylvania-New Jersey-Maryland Interconnect with the goal to identify patterns in the regulation signal that would be representative of the entire signal as a typical regulation data set. Results from an extensive time-series analysis are discussed, and the results are explained from both the statistical and the battery-testing perspectives. The results then are interpreted in the context of defining a small set of V2G drive cycles for standardization, offering some recommendations for the next steps toward standardizing testing protocols.

  17. The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in Bacillus cereus F4430/73▿

    PubMed Central

    Zigha, Assia; Rosenfeld, Eric; Schmitt, Philippe; Duport, Catherine

    2007-01-01

    Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O2 and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression. PMID:17259311

  18. A new regulator of pathogenicity (bvlR) is required for full virulence and tight microcolony formation in Pseudomonas aeruginosa.

    PubMed

    McCarthy, Ronan R; Mooij, Marlies J; Reen, F Jerry; Lesouhaitier, Olivier; O'Gara, Fergal

    2014-07-01

    LysR-type transcriptional regulators (LTTRs) are the most common family of transcriptional regulators found in the opportunistic pathogen Pseudomonas aeruginosa. They are known to regulate a wide variety of virulence determinants and have emerged recently as positive global regulators of pathogenicity in a broad spectrum of important bacterial pathogens. However, in spite of their key role in modulating expression of key virulence determinants underpinning pathogenic traits associated with the process of infection, surprisingly few are found to be transcriptionally altered by contact with host cells. BvlR (PA14_26880) an LTTR of previously unknown function, has been shown to be induced in response to host cell contact, and was therefore investigated for its potential role in virulence. BvlR expression was found to play a pivotal role in the regulation of acute virulence determinants such as type III secretion system and exotoxin A production. BvlR also played a key role in P. aeruginosa pathogenicity within the Caenorhabditis elegans acute model of infection. Loss of BvlR led to an inability to form tight microcolonies, a key step in biofilm formation in the cystic fibrosis lung, although surface attachment was increased. Unusually for LTTRs, BvlR was shown to exert its influence through the transcriptional repression of many genes, including the virulence-associated cupA and alg genes. This highlights the importance of BvlR as a new virulence regulator in P. aeruginosa with a central role in modulating key events in the pathogen-host interactome. PMID:24829363

  19. PtrA is required for coordinate regulation of gene expression during phosphate stress in a marine Synechococcus.

    PubMed

    Ostrowski, Martin; Mazard, Sophie; Tetu, Sasha G; Phillippy, Katherine; Johnson, Aaron; Palenik, Brian; Paulsen, Ian T; Scanlan, Dave J

    2010-07-01

    Previous microarray analyses have shown a key role for the two-component system PhoBR (SYNW0947, SYNW0948) in the regulation of P transport and metabolism in the marine cyanobacterium Synechococcus sp. WH8102. However, there is some evidence that another regulator, SYNW1019 (PtrA), probably under the control of PhoBR, is involved in the response to P depletion. PtrA is a member of the cAMP receptor protein transcriptional regulator family that shows homology to NtcA, the global nitrogen regulator in cyanobacteria. To define the role of this regulator, we constructed a mutant by insertional inactivation and compared the physiology of wild-type Synechcococcus sp. WH8102 with the ptrA mutant under P-replete and P-stress conditions. In response to P stress the ptrA mutant failed to upregulate phosphatase activity. Microarrays and quantitative RT-PCR indicate that a subset of the Pho regulon is controlled by PtrA, including two phosphatases, a predicted phytase and a gene of unknown function psip1 (SYNW0165), all of which are highly upregulated during P limitation. Electrophoretic mobility shift assays indicate binding of overexpressed PtrA to promoter sequences upstream of the induced genes. This work suggests a two-tiered response to P depletion in this strain, the first being PhoB-dependent induction of high-affinity PO(4) transporters, and the second the PtrA-dependent induction of phosphatases for scavenging organic P. The levels of numerous other transcripts are also directly or indirectly influenced by PtrA, including those involved in cell-surface modification, metal uptake, photosynthesis, stress responses and other metabolic processes, which may indicate a wider role for PtrA in cellular regulation in marine picocyanobacteria. PMID:20376102

  20. Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

    PubMed Central

    Cui, Cheng; Chatterjee, Bishwanath; Lozito, Thomas P.; Zhang, Zhen; Francis, Richard J.; Yagi, Hisato; Swanhart, Lisa M.; Sanker, Subramaniam; Francis, Deanne; Yu, Qing; San Agustin, Jovenal T.; Puligilla, Chandrakala; Chatterjee, Tania; Tansey, Terry; Liu, Xiaoqin; Kelley, Matthew W.; Spiliotis, Elias T.; Kwiatkowski, Adam V.; Tuan, Rocky; Pazour, Gregory J.; Hukriede, Neil A.; Lo, Cecilia W.

    2013-01-01

    Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to

  1. 31 CFR 103.120 - Anti-money laundering program requirements for financial institutions regulated by a Federal...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Anti-money laundering program... organization, and casinos. 103.120 Section 103.120 Money and Finance: Treasury Regulations Relating to Money and Finance FINANCIAL RECORDKEEPING AND REPORTING OF CURRENCY AND FOREIGN TRANSACTIONS...

  2. Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae

    PubMed Central

    Kaizer, Hannah; Connelly, Carla J.; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W.

    2015-01-01

    The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation. PMID:26294668

  3. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling.

    PubMed

    Fernandez-Alonso, R; Martin-Lopez, M; Gonzalez-Cano, L; Garcia, S; Castrillo, F; Diez-Prieto, I; Fernandez-Corona, A; Lorenzo-Marcos, M E; Li, X; Claesson-Welsh, L; Marques, M M; Marin, M C

    2015-08-01

    Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity. PMID:25571973

  4. Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions.

    PubMed

    Alexeeva, Svetlana; Hellingwerf, Klaas J; Teixeira de Mattos, M Joost

    2003-01-01

    In Escherichia coli, the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways. Previously, we have described the physiological response of wild-type E. coli to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 182:4934-4940, 2000, and S. Alexeeva, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 184:1402-1406, 2002). Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response. Wild-type E. coli and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis. A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states. Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions. In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome o- and d-terminal oxidases, and a significant change in the intracellular redox state were observed. Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator. PMID:12486057

  5. Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani.

    PubMed

    Vijayamahantesh; Amit, Ajay; Kumar, Santosh; Dikhit, Manas R; Jha, Pravin K; Singh, Ashish K; Sinha, Kislay K; Pandey, Krishna; Das, V N R; Das, Pradeep; Bimal, Sanjiva

    2016-03-01

    Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent. PMID:26748211

  6. The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization

    PubMed Central

    Azriel, Shalhevet; Goren, Alina; Rahav, Galia

    2015-01-01

    Salmonella enterica serovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide. Salmonella pathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that in S. Typhimurium, dksA is induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates the Salmonella pathogenicity island 1 and motility-chemotaxis genes and is necessary for S. Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable for S. Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found that dksA is spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infection in vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulence S. Typhimurium programs and therefore is a key virulence regulator of Salmonella. PMID:26553464

  7. The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization.

    PubMed

    Azriel, Shalhevet; Goren, Alina; Rahav, Galia; Gal-Mor, Ohad

    2016-01-01

    Salmonella enterica serovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide. Salmonella pathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that in S. Typhimurium, dksA is induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates the Salmonella pathogenicity island 1 and motility-chemotaxis genes and is necessary for S. Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable for S. Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found that dksA is spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infection in vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulence S. Typhimurium programs and therefore is a key virulence regulator of Salmonella. PMID:26553464

  8. The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans

    PubMed Central

    Han, Xue; Adames, Kelly; Sykes, Ellen M. E.; Srayko, Martin

    2015-01-01

    Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase. PMID:26168236

  9. Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae.

    PubMed Central

    Georgatsou, E; Alexandraki, D

    1994-01-01

    Iron uptake in Saccharomyces cerevisiae involves at least two steps: reduction of ferric to ferrous ions extracellularly and transport of the reduced ions through the plasma membrane. We have cloned and molecularly characterized FRE2, a gene which is shown to account, together with FRE1, for the total membrane-associated ferric reductase activity of the cell. Although not similar at the nucleotide level, the two genes encode proteins with significantly similar primary structures and very similar hydrophobicity profiles. The FRE1 and FRE2 proteins are functionally related, having comparable properties as ferric reductases. FRE2 expression, like FRE1 expression, is induced by iron deprivation, and at least part of this control takes place at the transcriptional level, since 156 nucleotides upstream of the initiator AUG conferred iron-dependent regulation when fused to a heterologous gene. However, the two gene products have distinct temporal regulation of their activities during cell growth. Images PMID:8164662

  10. RTN3 Regulates the Expression Level of Chemokine Receptor CXCR4 and is Required for Migration of Primordial Germ Cells

    PubMed Central

    Li, Haitao; Liang, Rong; Lu, Yanan; Wang, Mengxia; Li, Zandong

    2016-01-01

    CXCR4 is a crucial chemokine receptor that plays key roles in primordial germ cell (PGC) homing. To further characterize the CXCR4-mediated migration of PGCs, we screened CXCR4-interacting proteins using yeast two-hybrid screening. We identified reticulon3 (RTN3), a member of the reticulon family, and considered an apoptotic signal transducer, as able to interact directly with CXCR4. Furthermore, we discovered that the mRNA and protein expression levels of CXCR4 could be regulated by RTN3. We also found that RTN3 altered CXCR4 translocation and localization. Moreover, increasing the signaling of either CXCR4b or RTN3 produced similar PGC mislocalization phenotypes in zebrafish. These results suggested that RTN3 modulates PGC migration through interaction with, and regulation of, CXCR4. PMID:27070582

  11. Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

    PubMed Central

    Burbage, Marianne; Keppler, Selina J.; Gasparrini, Francesca; Martínez-Martín, Nuria; Gaya, Mauro; Feest, Christoph; Domart, Marie-Charlotte; Brakebusch, Cord; Collinson, Lucy; Bruckbauer, Andreas

    2015-01-01

    The small Rho GTPase Cdc42, known to interact with Wiskott–Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell–intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology. PMID:25547673

  12. Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

    PubMed Central

    Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

    2016-01-01

    Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

  13. Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

    PubMed Central

    Qu, Jing; Ero, Rya; Feng, Chen; Ong, Li-Teng; Tan, Hui-Foon; Lee, Hui-Shan; Ismail, Muhammad HB; Bu, Wen-Ting; Nama, Srikanth; Sampath, Prabha; Gao, Yong-Gui; Tan, Suet-Mien

    2015-01-01

    Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis. PMID:26677948

  14. The Yeast Prion [SWI(+)] Abolishes Multicellular Growth by Triggering Conformational Changes of Multiple Regulators Required for Flocculin Gene Expression.

    PubMed

    Du, Zhiqiang; Zhang, Ying; Li, Liming

    2015-12-29

    Although transcription factors are prevalent among yeast prion proteins, the role of prion-mediated transcriptional regulation remains elusive. Here, we show that the yeast prion [SWI(+)] abolishes flocculin (FLO) gene expression and results in a complete loss of multicellularity. Further investigation demonstrates that besides Swi1, multiple other proteins essential for FLO expression, including Mss11, Sap30, and Msn1 also undergo conformational changes and become inactivated in [SWI(+)] cells. Moreover, the asparagine-rich region of Mss11 can exist as prion-like aggregates specifically in [SWI(+)] cells, which are SDS resistant, heritable, and curable, but become metastable after separation from [SWI(+)]. Our findings thus reveal a prion-mediated mechanism through which multiple regulators in a biological pathway can be inactivated. In combination with the partial loss-of-function phenotypes of [SWI(+)] cells on non-glucose sugar utilization, our data therefore demonstrate that a prion can influence distinct traits differently through multi-level regulations, providing insights into the biological roles of prions. PMID:26711350

  15. Cartilage development requires the function of Estrogen-related receptor alpha that directly regulates sox9 expression in zebrafish

    PubMed Central

    Kim, Yong-Il; No Lee, Joon; Bhandari, Sushil; Nam, In-Koo; Yoo, Kyeong-Won; Kim, Se-Jin; Oh, Gi-Su; Kim, Hyung-Jin; So, Hong-Seob; Choe, Seong-Kyu; Park, Raekil

    2015-01-01

    Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development. PMID:26657540

  16. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.

    PubMed

    Zhao, Xu; Yang, Ying; Sun, Bao-Fa; Shi, Yue; Yang, Xin; Xiao, Wen; Hao, Ya-Juan; Ping, Xiao-Li; Chen, Yu-Sheng; Wang, Wen-Jia; Jin, Kang-Xuan; Wang, Xing; Huang, Chun-Min; Fu, Yu; Ge, Xiao-Meng; Song, Shu-Hui; Jeong, Hyun Seok; Yanagisawa, Hiroyuki; Niu, Yamei; Jia, Gui-Fang; Wu, Wei; Tong, Wei-Min; Okamoto, Akimitsu; He, Chuan; Rendtlew Danielsen, Jannie M; Wang, Xiu-Jie; Yang, Yun-Gui

    2014-12-01

    The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis. PMID:25412662

  17. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis

    PubMed Central

    Zhao, Xu; Yang, Ying; Sun, Bao-Fa; Shi, Yue; Yang, Xin; Xiao, Wen; Hao, Ya-Juan; Ping, Xiao-Li; Chen, Yu-Sheng; Wang, Wen-Jia; Jin, Kang-Xuan; Wang, Xing; Huang, Chun-Min; Fu, Yu; Ge, Xiao-Meng; Song, Shu-Hui; Jeong, Hyun Seok; Yanagisawa, Hiroyuki; Niu, Yamei; Jia, Gui-Fang; Wu, Wei; Tong, Wei-Min; Okamoto, Akimitsu; He, Chuan; Danielsen, Jannie M Rendtlew; Wang, Xiu-Jie; Yang, Yun-Gui

    2014-01-01

    The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis. PMID:25412662

  18. The yeast prion [SWI+] abolishes multicellular growth by triggering conformational changes of multiple regulators required for flocculin gene expression

    PubMed Central

    Du, Zhiqiang; Zhang, Ying; Li, Liming

    2016-01-01

    Summary While transcription factors are prevalent among yeast prion proteins, the role of prion-mediated transcriptional regulation remains elusive. We show here that the yeast prion [SWI+] abolishes flocculin (FLO) gene expression and results in a complete loss of multicellularity. Further investigation demonstrates that besides Swi1, multiple other proteins essential for FLO expression, including Mss11, Sap30, and Msn1 also undergo conformational changes, and become inactivated in [SWI+] cells. Moreover, the asparagine-rich region of Mss11 can exist as prion-like aggregates specifically in [SWI+] cells, which are SDS-resistant, heritable, and curable, but become metastable after separation from [SWI+]. Our findings thus reveal a prion-mediated mechanism through which multiple regulators in a biological pathway can be inactivated. In combination with the partial loss-of-function phenotypes of [SWI+] cells on non-glucose sugar utilization, our data therefore demonstrate that a prion can influence differently on distinct traits through multi-level regulations, providing insights into the biological roles of prions. PMID:26711350

  19. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production

    PubMed Central

    Chen, Gang; Korfhagen, Thomas R.; Xu, Yan; Kitzmiller, Joseph; Wert, Susan E.; Maeda, Yutaka; Gregorieff, Alexander; Clevers, Hans; Whitsett, Jeffrey A.

    2009-01-01

    Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain–containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage–tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders. PMID:19759516

  20. Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii.

    PubMed

    Oda, Saori; Yurimoto, Hiroya; Nitta, Nobuhisa; Sakai, Yasuyoshi

    2016-05-01

    The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (Δ256-292 or Δ107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (Δ1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3Δ strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3Δ strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA. PMID:26963751

  1. A peroxiredoxin, PRDX-2, is required for insulin secretion and insulin/IIS-dependent regulation of stress resistance and longevity.

    PubMed

    Oláhová, Monika; Veal, Elizabeth A

    2015-08-01

    Peroxiredoxins (Prx) are abundant thiol peroxidases with a conserved anti-ageing role. In contrast to most animals, the nematode worm, Caenorhabditis elegans, encodes a single cytosolic 2-Cys Prx, PRDX-2, rendering it an excellent model for examining how peroxiredoxins affect animal physiology and ageing. Our previous work revealed that, although PRDX-2 protects against the toxicity of peroxides, enigmatically, prdx-2-mutant animals are hyper-resistant to other forms of oxidative stress. Here, we have investigated the basis for this increased resistance. Mammalian FOXO and Nrf2 transcription factors directly promote the expression of a range of detoxification enzymes. We show that the FOXO orthologue, DAF-16, and the Nrf2 orthologue, SKN-1, are required for the increased stress resistance of prdx-2-mutant worms. Our data suggest that PRDX-2 is required for normal levels of insulin secretion and hence the inhibition of DAF-16 and SKN-1 by insulin/IGF-1-like signalling (IIS) under nutrient-rich conditions. Intriguingly, loss of PRDX-2 increases DAF-16 and SKN-1 activities sufficiently to increase arsenite resistance without initiating other IIS-inhibited processes. Together, these data suggest that loss of peroxiredoxin function may increase stress resistance by reducing insulin secretion, but that further changes in insulin signalling are required for the reprogramming of development and fat metabolism. In addition, we reveal that the temperature-dependent prolongevity function of PRDX-2 is required for the extended lifespan associated with several pathways, including further reductions in IIS. PMID:25808059

  2. A DNA2 Homolog Is Required for DNA Damage Repair, Cell Cycle Regulation, and Meristem Maintenance in Plants1[OPEN

    PubMed Central

    Jia, Ning; Liu, Xiaomin; Gao, Hongbo

    2016-01-01

    Plant meristem cells divide and differentiate in a spatially and temporally regulated manner, ultimately giving rise to organs. In this study, we isolated the Arabidopsis jing he sheng 1 (jhs1) mutant, which exhibited retarded growth, an abnormal pattern of meristem cell division and differentiation, and morphological defects such as fasciation, an irregular arrangement of siliques, and short roots. We identified JHS1 as a homolog of human and yeast DNA Replication Helicase/Nuclease2, which is known to be involved in DNA replication and damage repair. JHS1 is strongly expressed in the meristem of Arabidopsis. The jhs1 mutant was sensitive to DNA damage stress and had an increased DNA damage response, including increased expression of genes involved in DNA damage repair and cell cycle regulation, and a higher frequency of homologous recombination. In the meristem of the mutant plants, cell cycle progression was delayed at the G2 or late S phase and genes essential for meristem maintenance were misregulated. These results suggest that JHS1 plays an important role in DNA replication and damage repair, meristem maintenance, and development in plants. PMID:26951435

  3. Expression atlas of the multivalent epigenetic regulator Brpf1 and its requirement for survival of mouse embryos.

    PubMed

    You, Linya; Chen, Lulu; Penney, Janice; Miao, Dengshun; Yang, Xiang-Jiao

    2014-06-01

    Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a unique epigenetic regulator that contains multiple structural domains for recognizing different chromatin modifications. In addition, it possesses sequence motifs for forming multiple complexes with three different histone acetyltransferases, MOZ, MORF, and HBO1. Within these complexes, BRPF1 serves as a scaffold for bridging subunit interaction, stimulating acetyltransferase activity, governing substrate specificity and stimulating gene expression. To investigate how these molecular interactions are extrapolated to biological functions of BRPF1, we utilized a mouse strain containing a knock-in reporter and analyzed the spatiotemporal expression from embryos to adults. The analysis revealed dynamic expression in the extraembryonic, embryonic, and fetal tissues, suggesting important roles of Brpf1 in prenatal development. In support of this, inactivation of the mouse Brpf1 gene causes lethality around embryonic day 9.5. After birth, high expression is present in the testis and specific regions of the brain. The 4-dimensional expression atlas of mouse Brpf1 should serve as a valuable guide for analyzing its interaction with Moz, Morf, and Hbo1 in vivo, as well as for investigating whether Brpf1 functions independently of these three enzymatic epigenetic regulators. PMID:24646517

  4. SORTING NEXIN1 is required for modulating the trafficking and stability of the Arabidopsis IRON-REGULATED TRANSPORTER1.

    PubMed

    Ivanov, Rumen; Brumbarova, Tzvetina; Blum, Ailisa; Jantke, Anna-Maria; Fink-Straube, Claudia; Bauer, Petra

    2014-03-01

    Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery. PMID:24596241

  5. Expression atlas of the multivalent epigenetic regulator Brpf1 and its requirement for survival of mouse embryos

    PubMed Central

    You, Linya; Chen, Lulu; Penney, Janice; Miao, Dengshun; Yang, Xiang-Jiao

    2014-01-01

    Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a unique epigenetic regulator that contains multiple structural domains for recognizing different chromatin modifications. In addition, it possesses sequence motifs for forming multiple complexes with three different histone acetyltransferases, MOZ, MORF, and HBO1. Within these complexes, BRPF1 serves as a scaffold for bridging subunit interaction, stimulating acetyltransferase activity, governing substrate specificity and stimulating gene expression. To investigate how these molecular interactions are extrapolated to biological functions of BRPF1, we utilized a mouse strain containing a knock-in reporter and analyzed the spatiotemporal expression from embryos to adults. The analysis revealed dynamic expression in the extraembryonic, embryonic, and fetal tissues, suggesting important roles of Brpf1 in prenatal development. In support of this, inactivation of the mouse Brpf1 gene causes lethality around embryonic day 9.5. After birth, high expression is present in the testis and specific regions of the brain. The 4-dimensional expression atlas of mouse Brpf1 should serve as a valuable guide for analyzing its interaction with Moz, Morf, and Hbo1 in vivo, as well as for investigating whether Brpf1 functions independently of these three enzymatic epigenetic regulators. PMID:24646517

  6. Regulatory Requirements and Technical Analysis for Department of Energy Regulated Performance Assessments of Shallow-Trench Disposal of Low-Level Radioactive Waste at the Nevada Test Site

    NASA Astrophysics Data System (ADS)

    Crowe, B.; Black, P.; Tauxe, J.; Yucel, V.; Rawlinson, S.; Colarusso, A.; DiSanza, F.

    2001-12-01

    The National Nuclear Security Administration, Nevada Operations Office (NNSA/NV) operates and maintains two active facilities on the Nevada Test Site (NTS) that dispose Department of Energy (DOE) defense-generated low-level radioactive (LLW), mixed radioactive, and classified waste in shallow trenches, pits and large-diameter boreholes. The operation and maintenance of the LLW disposal sites are self-regulated under DOE Order 435.1, which requires review of a Performance Assessment for four performance objectives: 1) all pathways 25 mrem/yr limit; 2) atmospheric pathways 10 mrem/yr limit; 3) radon flux density of 20 pCi/m2/s; and 4) groundwater resource protection (Safe Drinking Water Act; 4 mrem/yr limit). The inadvertent human intruder is protected under a dual 500- and 100-mrem limit (acute and chronic exposure). In response to the Defense Nuclear Facilities Safety Board Recommendation 92 2, a composite analysis is required that must examine all interacting sources for compliance against both 30 and 100 mrem/yr limits. A small component of classified transuranic waste is buried at intermediate depths in 3-meter diameter boreholes at the Area 5 LLW disposal facility and is assessed through DOE-agreement against the requirements of the Environmental Protection Agency (EPA)'s 40 CFR 191. The hazardous components of mixed LLW are assessed against RCRA requirements. The NTS LLW sites fall directly under three sets of federal regulations and the regulatory differences result not only in organizational challenges, but also in different decision objectives and technical paths to completion. The DOE regulations require deterministic analysis for a 1,000-year compliance assessment supplemented by probabilistic analysis under a long-term maintenance program. The EPA regulations for TRU waste are probabilistically based for a compliance interval of 10,000 years. Multiple steps in the assessments are strongly dependent on assumptions for long-term land use policies

  7. Na/sup +/ requirement for growth, photosynthesis, and pH regulation of the alkalotolerant cyanobacterium Synechococcus leopoliensis

    SciTech Connect

    Miller, A.G.; Turpin, D.H.; Canvin, D.T.

    1984-07-01

    It was found that Na/sup +/ is required for all the alkalotolerance of the cyanobacterium Synechococcus leopoliensis. Cell division did not occur at any pH in the absence of Na/sup +/, but cells inoculated into Na/sup +/-free growth medium at pH 6.8 did continue metabolic activity, and over a period of 48 h, the cells became twice their normal size. Many of these cells remained viable for at least 59 h and formed colonies on Na/sup +/-containing medium. Cells grown in the presence of Na/sup +/ and inoculated into Na/sup +/-free growth medium at pH 9.6 rapidly lost viability. An Na/sup +/ concentration of ca. 0.5 milliequivalents x liter/sup -1/ was required for sustained growth above pH 9.0. The Na/sup +/ requirement could be only partially met by Li/sup +/ and not at all by K/sup +/ or Rb/sup +/. Cells incubated in darkness in growth medium at pH 6.8 had an intracellular pH near neutrality in the presence or absence of Na/sup +/. When the external pH was shifted to 9.6, only cells in the presence of Na/sup +/ were able to maintain an intracellular pH near 7.0. The membrane potential, however, remained high (-120mV) in the absence or presence of Na/sup +/ unless collapsed by the addition of gramicidin. Thus, the inability to maintain a neutral intracellular pH at pH 9.6 in the absence of Na/sup +/ was not due to a generalized disruption of membrane integrity. Even cells containing Na/sup +/ still required added Na/sup +/ to restore photosynthetic rates to normal after the cells had been washed in Na/sup +/-free buffer at pH 9.6. This requirement was only partially met by Li/sup +/ and was not met at all by K/sup +/, Rb/sup +/, Cs/sup +/, Mg/sup 2 +/, or Ca/sup 2 +/. The restoration of photosynthesis by added Na/sup +/ occurred within 30 s and suggests a role for extracellular Na/sup +/. Part of our results can be explained in terms of the operation of an Na/sup +//H/sup +/ antiporter activity in the plasma membrane, but some results would seem to require other

  8. StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration.

    PubMed

    Gu, Shou-Qin; Li, Po; Wu, Min; Hao, Zhi-Min; Gong, Xiao-Dong; Zhang, Xiao-Yu; Tian, Lan; Zhang, Pan; Wang, Yue; Cao, Zhi-Yan; Fan, Yong-Shan; Han, Jian-Min; Dong, Jin-Gao

    2014-11-01

    In filamentous fungi, the pathogenic mitogen-activated protein kinase (PMK) pathway performs an important function in plant infection. STE12-like genes found in higher eukaryotes encode transcription factors and are regulated by the PMK pathway. However, the functions of STE12-like genes in foliar pathogens remain poorly understood. In this study, we cloned StSTE12 from Setosphaeria turcica and investigated its functions by RNA interference. Transformants ste12-3, ste12-2 and, ste12-1, in which the StSTE12 silencing efficiency increased in order, were confirmed by real time PCR. Compared with the wild-type (WT) strain, the transformants showed reduced growth rate, lighter colony color, and obviously decreased conidium production. More importantly, different to WT strain and ste12-3 with lower StSTE12silencing efficiency, ste12-1 and ste12-2 with higher StSTE12 silencing efficiency were nonpathogenic on intact leaves, but pathogenic on wounded leaves. However, the biological activity of HT-toxin from all transformants showed no difference on corn leaves. Furthermore, ste12-1 and ste12-2 did not penetrate artificial cellophane membrane and showed abnormal and delayed development appressoria. Although it could penetrate the cellophane membranes, ste12-3 formed appressoria after 48 h of inoculation more than WT. Therefore, StSTE12 was involved in vegetative growth, conidiation, appressorial development, penetration as well as the pathogenicity, but it was not related to HT-toxin biosynthesis. More interestingly, all the results suggested that StSTE12 was crucial for pathogenicity due to involvement in regulating appressoria development and penetration. PMID:24813304

  9. Structure of a Highly Conserved Domain of Rock1 Required for Shroom-Mediated Regulation of Cell Morphology

    PubMed Central

    Bauer, Robert J.; Heroux, Annie; Zalewski, Jenna K.; Heber, Simone; Dosunmu-Ogunbi, Atinuke M.; Trakselis, Michael A.; Hildebrand, Jeffrey D.; VanDemark, Andrew P.

    2013-01-01

    Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology. PMID:24349032

  10. A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5*

    PubMed Central

    Vild, Cody J.; Li, Yan; Guo, Emily Z.; Liu, Yuan; Xu, Zhaohui

    2015-01-01

    Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. Here, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct from what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr182) at the core of LIP5NTD structure. Mutation of Tyr182 partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4. PMID:25637630

  11. Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

    PubMed

    Resetár, Anna; Demeter, Zita; Ficsor, Emese; Balázs, Andrea; Mosolygó, Agnes; Szőke, Eva; Gonda, S; Papp, L; Surányi, G; Máthé, C

    2014-06-01

    In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids. PMID:24873910

  12. Xanthomonas oryzae pv. oryzae requires H-NS-family protein XrvC to regulate virulence during rice infection.

    PubMed

    Liu, Yongting; Long, Juying; Shen, Dan; Song, Congfeng

    2016-05-01

    Histone-like nucleoid-structuring (H-NS) proteins, which are conserved in Gram-negative bacteria, bind DNA and act as the global transcriptional repressors. In this study, we identified and characterized the xrvC gene encoding a H-NS protein in Xathomonas oryzae pv. oryzae (Xoo) Philippines strain PXO99(A) Compared with the wild type, the xrvC-deficient mutant of PXO99(A) (named PXO99ΔxrvC) showed a reduced growth rate in both nutrient-rich and nutrient-limited media. Interestingly, PXO99ΔxrvC exhibited significantly reduced virulence on rice cultivar IRBB214, but its virulence on 31 other rice cultivars was not affected. Transcriptional analysis revealed that the expression of hrpG, hrpX and hpa1 and of 15 out of 18 tested non-TAL (transcription activator-like) effector genes was decreased significantly in the xrvC mutant compared with that in the wild type. In addition, loss of xrvC also impaired the induction of the rice susceptibility gene Os8N3 in IRBB214 by PXO99(A) Our results suggest that the xrvC gene is involved in bacterial growth, and it plays a vital role in virulence by positively regulating the expression of hrp genes and non-TAL effector genes in PXO99(A) and the susceptibility gene Os8N3 in rice. PMID:27001973

  13. The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage.

    PubMed

    Lindley, Linsey E; Curtis, Kevin M; Sanchez-Mejias, Avencia; Rieger, Megan E; Robbins, David J; Briegel, Karoline J

    2015-03-01

    The identification of multipotent mammary stem cells (MaSCs) has provided an explanation for the unique regenerative capacity of the mammary gland throughout adult life. However, it remains unclear what genes maintain MaSCs and control their specification into the two epithelial lineages: luminal and basal. LBH is a novel transcription co-factor in the WNT pathway with hitherto unknown physiological function. LBH is expressed during mammary gland development and aberrantly overexpressed in aggressive 'basal' subtype breast cancers. Here, we have explored the in vivo role of LBH in mammopoiesis. We show that in postnatal mammary epithelia, LBH is predominantly expressed in the Lin(-)CD29(high)CD24(+) basal MaSC population. Upon conditional inactivation of LBH, mice exhibit pronounced delays in mammary tissue expansion during puberty and pregnancy, accompanied by increased luminal differentiation at the expense of basal lineage specification. These defects could be traced to a severe reduction in the frequency and self-renewal/differentiation potential of basal MaSCs. Mechanistically, LBH induces expression of key epithelial stem cell transcription factor ΔNp63 to promote a basal MaSC state and repress luminal differentiation genes, mainly that encoding estrogen receptor α (Esr1/ERα). Collectively, these studies identify LBH as an essential regulator of basal MaSC expansion/maintenance, raising important implications for its potential role in breast cancer pathogenesis. PMID:25655704

  14. Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.

    PubMed

    Luis, Nuno Miguel; Morey, Lluis; Mejetta, Stefania; Pascual, Gloria; Janich, Peggy; Kuebler, Bernd; Cozutto, Luca; Roma, Guglielmo; Nascimento, Elisabete; Frye, Michaela; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-09-01

    Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. PMID:21885019

  15. Mitogen-activated protein kinase cascade required for regulation of development and secondary metabolism in Neurospora crassa.

    PubMed

    Park, Gyungsoon; Pan, Songqin; Borkovich, Katherine A

    2008-12-01

    Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi. PMID:18849472

  16. The bristle patterning genes hairy and extramacrochaetae regulate the development of structures required for flight in Diptera☆

    PubMed Central

    Costa, Marta; Calleja, Manuel; Alonso, Claudio R.; Simpson, Pat

    2014-01-01

    The distribution of sensory bristles on the thorax of Diptera (true flies) provides a useful model for the study of the evolution of spatial patterns. Large bristles called macrochaetes are arranged into species-specific stereotypical patterns determined via spatially discrete expression of the proneural genes achaete–scute (ac–sc). In Drosophila ac-sc expression is regulated by transcriptional activation at sites where bristle precursors develop and by repression outside of these sites. Three genes, extramacrochaetae (emc), hairy (h) and stripe (sr), involved in repression have been documented. Here we demonstrate that in Drosophila, the repressor genes emc and h, like sr, play an essential role in the development of structures forming part of the flight apparatus. In addition we find that, in Calliphora vicina a species diverged from D. melanogaster by about 100 Myr, spatial expression of emc, h and sr is conserved at the location of development of those structures. Based on these findings we argue, first, that the role emc, h and sr in development of the flight apparatus preceded their activities for macrochaete patterning; second, that species-specific variation in activation and repression of ac-sc expression is evolving in parallel to establish a unique distribution of macrochaetes in each species. PMID:24384389

  17. Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling

    PubMed Central

    Zhang, Zilai; Cao, Mou; Chang, Chia-Wei; Wang, Cindy; Shi, Xuanming; Zhan, Xiaoming; Birnbaum, Shari G.; Bezprozvanny, Ilya; Huber, Kimberly M.

    2015-01-01

    Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD. PMID:26459759

  18. Extinction of aversive memories associated with morphine withdrawal requires ERK-mediated epigenetic regulation of brain-derived neurotrophic factor transcription in the rat ventromedial prefrontal cortex.

    PubMed

    Wang, Wei-Sheng; Kang, Shuo; Liu, Wen-Tao; Li, Mu; Liu, Yao; Yu, Chuan; Chen, Jie; Chi, Zhi-Qiang; He, Ling; Liu, Jing-Gen

    2012-10-01

    Recent evidence suggests that histone deacetylase (HDAC) inhibitors facilitate extinction of rewarding memory of drug taking. However, little is known about the role of chromatin modification in the extinction of aversive memory of drug withdrawal. In this study, we used conditioned place aversion (CPA), a highly sensitive model for measuring aversive memory of drug withdrawal, to investigate the role of epigenetic regulation of brain-derived neurotrophic factor (BDNF) gene expression in extinction of aversive memory. We found that CPA extinction training induced an increase in recruiting cAMP response element-binding protein (CREB) to and acetylation of histone H3 at the promoters of BDNF exon I transcript and increased BDNF mRNA and protein expression in the ventromedial prefrontal cortex (vmPFC) of acute morphine-dependent rats and that such epigenetic regulation of BDNF gene transcription could be facilitated or diminished by intra-vmPFC infusion of HDAC inhibitor trichostatin A or extracellular signal-regulated kinase (ERK) inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene) before extinction training. Correspondingly, disruption of the epigenetic regulation of BDNF gene transcription with U0126 or suppression of BDNF signaling with Trk receptor antagonist K252a or BDNF scavenger tyrosine kinase receptor B (TrkB)-Fc blocked extinction of CPA behavior. We also found that extinction training-induced activation of ERK and CREB and extinction of CPA behavior could be potentiated or suppressed by intra-vmPFC infusion of d-cycloserine, a NMDA receptor partial agonist or aminophosphonopentanoic acid, a NMDA receptor antagonist. We conclude that extinction of aversive memory of morphine withdrawal requires epigenetic regulation of BDNF gene transcription in the vmPFC through activation of the ERK-CREB signaling pathway perhaps in a NMDA receptor-dependent manner. PMID:23035088

  19. Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

    PubMed Central

    Davy, Alice; Gale, Nicholas W.; Murray, Elizabeth W.; Klinghoffer, Richard A.; Soriano, Philippe; Feuerstein, Claude; Robbins, Stephen M.

    1999-01-01

    Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development. PMID:10601038

  20. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

    PubMed

    Liu, Jessica A J; Wu, Ming-Hoi; Yan, Carol H; Chau, Bolton K H; So, Henry; Ng, Alvis; Chan, Alan; Cheah, Kathryn S E; Briscoe, James; Cheung, Martin

    2013-02-19

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

  1. Crimean-Congo Hemorrhagic Fever Virus Entry into Host Cells Occurs through the Multivesicular Body and Requires ESCRT Regulators

    PubMed Central

    Shtanko, Olena; Nikitina, Raisa A.; Altuntas, Cengiz Z.; Chepurnov, Alexander A.; Davey, Robert A.

    2014-01-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus causing outbreaks of severe disease in humans, with a fatality rate approaching 30%. There are no widely accepted therapeutics available to prevent or treat the disease. CCHFV enters host cells through clathrin-mediated endocytosis and is subsequently transported to an acidified compartment where the fusion of virus envelope with cellular membranes takes place. To better understand the uptake pathway, we sought to identify host factors controlling CCHFV transport through the cell. We demonstrate that after passing through early endosomes in a Rab5-dependent manner, CCHFV is delivered to multivesicular bodies (MVBs). Virus particles localized to MVBs approximately 1 hour after infection and affected the distribution of the organelle within cells. Interestingly, blocking Rab7 activity had no effect on association of the virus with MVBs. Productive virus infection depended on phosphatidylinositol 3-kinase (PI3K) activity, which meditates the formation of functional MVBs. Silencing Tsg101, Vps24, Vps4B, or Alix/Aip1, components of the endosomal sorting complex required for transport (ESCRT) pathway controlling MVB biogenesis, inhibited infection of wild-type virus as well as a novel pseudotyped vesicular stomatitis virus (VSV) bearing CCHFV glycoprotein, supporting a role for the MVB pathway in CCHFV entry. We further demonstrate that blocking transport out of MVBs still allowed virus entry while preventing vesicular acidification, required for membrane fusion, trapped virions in the MVBs. These findings suggest that MVBs are necessary for infection and are the sites of virus-endosome membrane fusion. PMID:25233119

  2. Proliferating Cell Nuclear Antigen (PCNA) Is Required for Cell Cycle-regulated Silent Chromatin on Replicated and Nonreplicated Genes*

    PubMed Central

    Miller, Andrew; Chen, Jiji; Takasuka, Taichi E.; Jacobi, Jennifer L.; Kaufman, Paul D.; Irudayaraj, Joseph M. K.; Kirchmaier, Ann L.

    2010-01-01

    In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793–809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications. PMID:20813847

  3. Palmitoylation on the carboxyl terminus tail is required for the selective regulation of dopamine D2 versus D3 receptors.

    PubMed

    Zhang, Xiaowei; Le, Hang Thi; Zhang, Xiaohan; Zheng, Mei; Choi, Bo-Gil; Kim, Kyeong-Man

    2016-09-01

    Dopamine D2 receptor (D2R) and D3 receptor (D3R) possess highly conserved amino acid sequences but this study showed that D3R was more extensively palmitoylated than D2R. Based on this finding, the molecular basis of this selective palmitoylation of D3R was determined and the roles of palmitoylation in the regulation of D3R functions were investigated. D3R was palmitoylated on the cysteine residue on its carboxyl terminus tail, the last amino acid residue of D3R, and an exchange of the carboxyl terminus tail between D2R and D3R (D2R-D3C and D3R-D2C) resulted in the switching of the palmitoylation phenotype. When the consensus site for palmitoylation was mutated or the palmitoylation of D3R was inhibited by treatment with 2-bromopalmitate (2BP), a palmitoylation blocker, cell-surface expression, PKC-mediated endocytosis, agonist affinity, and agonist-induced tolerance of D3R were all inhibited. However, these changes were not observed when D3R palmitoylation was inhibited by replacing its carboxyl tail with that of D2R (D3R-D2C) or when the palmitoylation of D2R-D3C was inhibited by treatment with 2BP. Overall, this study shows that D3R is palmitoylated more extensively than D2R even though the carboxyl terminus tails of D2R and D3R are highly homologous, and thus provides a new clue regarding the consensus sequence for palmitoylation. This study also shows that palmitoylation controls various functionalities of D3R only when the receptor is in the intact D3R configuration. PMID:27349735

  4. Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa.

    PubMed

    Serino, L; Reimmann, C; Visca, P; Beyeler, M; Chiesa, V D; Haas, D

    1997-01-01

    The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron

  5. NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation.

    PubMed

    Yang, Meixiang; Chen, Shasha; Du, Juan; He, Junming; Wang, Yuande; Li, Zehua; Liu, Guangao; Peng, Wanwen; Zeng, Xiaokang; Li, Dan; Xu, Panglian; Guo, Wei; Chang, Zai; Wang, Song; Tian, Zhigang; Dong, Zhongjun

    2016-01-01

    Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of 'missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development. PMID:27601261

  6. Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation.

    PubMed Central

    Sommer, J M; Newton, A

    1988-01-01

    Pili, along with the flagellum and DNA bacteriophage receptors, are structural markers for polar morphogenesis in Caulobacter crescentus. Pili act as primary receptors for a number of small, C. crescentus-specific DNA and RNA bacteriophages, and the timing of pilus-dependent adsorption of bacteriophage phiCb5 in synchronized cell populations has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisional cell. The use of rotary platinum shadow casting and electron microscopy as a direct assay for formation of flagella and pili in synchronous cell cultures now shows, however, that when expressed as fractions of the swarmer cell cycle, flagella are assembled on the predivisional cells at approximately 0.8 and that pili are assembled on the new swarmer cells at approximately 0.1 of the next cell cycle. Adsorption of pilus-specific bacteriophage phiCb5 prevented the loss of pili from swarmer cells during development, which suggests that these structures are retracted at the time of stalk formation. Examination of temperature-sensitive cell division mutants showed that the assembly of pili depends on completion of cell separation. These results indicate that the stage-specific events required for polar morphogenesis in C. crescentus occur sequentially, rather than coordinately in the cell cycle, and that the timing of these events reflects the order of underlying cell cycle steps. Images PMID:2891681

  7. Synthesis of fusogenic lipids through activation of phospholipase D1 by GTPases and the kinase RSK2 is required for calcium-regulated exocytosis in neuroendocrine cells.

    PubMed

    Vitale, Nicolas

    2010-02-01

    Exocytosis of hormones occurs through the fusion of large dense-core secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and also specific lipids at the site of membrane fusion. Among the different lipids required for exocytosis, our recent observations have highlighted the crucial role of PA (phosphatidic acid) in the late stages of membrane fusion in various exocytotic events. An RNAi (RNA interference) strategy coupled with the detection of PA in living cells has pointed to plasma membrane-associated PLD1 (phospholipase D(1)) as the main producer of PA in response to secretagogue stimulation. We have identified several GTPases which regulate the activation level of PLD(1) in neuroendocrine cells. Finally, RSK2 (ribosomal S6 kinase 2) appears to phosphorylate and regulate the activity of PLD(1) in a calcium-dependent manner. Altogether our results have unravelled a complex set of regulatory pathways controlling the synthesis of fusogenic lipids at the secretory granule fusion site by PLD(1). PMID:20074053

  8. NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development.

    PubMed

    Sanchez-Ortiz, Efrain; Cho, Woosung; Nazarenko, Inga; Mo, Wei; Chen, Jian; Parada, Luis F

    2014-11-01

    Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders. PMID:25367036

  9. Post-developmental microRNA expression is required for normal physiology, and regulates aging in parallel to insulin/IGF-1 signaling in C. elegans.

    PubMed

    Lehrbach, Nicolas J; Castro, Cecilia; Murfitt, Kenneth J; Abreu-Goodger, Cei; Griffin, Julian L; Miska, Eric A

    2012-12-01

    Regulation of gene expression by microRNAs (miRNAs) is essential for normal development, but the roles of miRNAs in the physiology of adult animals are poorly understood. We have isolated a conditional allele of DGCR8/pash-1, which allows reversible and rapid inactivation of miRNA synthesis in vivo in Caenorhabditis elegans. This is a powerful new tool that allows dissection of post-developmental miRNA functions. We demonstrate that continuous synthesis of miRNAs is dispensable for cellular viability but critical for the physiology of adult animals. Loss of miRNA synthesis in the adult reduces lifespan and results in rapid aging. The insulin/IGF-1 signaling pathway is a critical determinant of lifespan, and is modulated by miRNAs. We find that although miRNA expression is required for some mechanisms of lifespan extension, it is not essential for the longevity of animals lacking insulin/IGF-1 signaling. Further, misregulated insulin/IGF-1 signaling cannot account for the reduced lifespan caused by disruption of miRNA synthesis. We show that miRNAs act in parallel with insulin/IGF-1 signaling to regulate a shared set of downstream genes important for physiological processes that determine lifespan. We conclude that coordinated transcriptional and post-transcriptional regulation of gene expression promotes longevity. PMID:23097426

  10. Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression

    PubMed Central

    Fink, Karin; Martel, Alexis; Jouan, Loubna; Nzengue, Yves; Lamarre, Daniel; Vande Velde, Christine; Grandvaux, Nathalie

    2010-01-01

    The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNβ and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS. PMID:20532218

  11. Nitrogen regulation of nasA and the nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis.

    PubMed Central

    Nakano, M M; Yang, F; Hardin, P; Zuber, P

    1995-01-01

    The divergently transcribed nasA gene and nasB operon are required for nitrate and nitrite assimilation in Bacillus subtilis. The beta-galactosidase activity of transcriptional lacZ fusions from the nasA and nasB promoters was high when cells were grown in minimal glucose medium containing poor nitrogen sources such as nitrate, proline, or glutamate. The expression was very low when ammonium or glutamine was used as the sole nitrogen source. The repression of the genes during growth on good sources of nitrogen required wild-type glutamine synthetase (GlnA), but not GlnR, the repressor of the glnRA operon. Primer extension analysis showed that the -10 region of each promoter resembles those of sigma A-recognized promoters. Between the divergently oriented nasA and nasB promoters is a region of dyad symmetry. Mutational analysis led to the conclusion that this sequence is required in cis for the activation of both nasA and nasB. The derepression of these genes in a glnA mutant also required this sequence. These results suggest that an unidentified transcriptional activator and glutamine synthetase function in the regulation of nasA and the nasB operon. PMID:7836289

  12. Requirements for Foreign and Domestic Establishment Registration and Listing for Human Drugs, Including Drugs That Are Regulated Under a Biologics License Application, and Animal Drugs. Final rule.

    PubMed

    2016-08-31

    The Food and Drug Administration (FDA) is amending its regulations governing drug establishment registration and drug listing. These amendments reorganize, modify, and clarify current regulations concerning who must register establishments and list human drugs, human drugs that are also biological products, and animal drugs. The final rule requires electronic submission, unless waived in certain circumstances, of registration and listing information. This rulemaking pertains to finished drug products and to active pharmaceutical ingredients (APIs) alone or together with one or more other ingredients. The final rule describes how and when owners or operators of establishments at which drugs are manufactured or processed must register their establishments with FDA and list the drugs they manufacture or process. In addition, the rule makes certain changes to the National Drug Code (NDC) system. We are taking this action to improve management of drug establishment registration and drug listing requirements and make these processes more efficient and effective for industry and for us. This action also supports implementation of the electronic prescribing provisions of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) and the availability of current drug labeling information through DailyMed, a computerized repository of drug information maintained by the National Library of Medicine. PMID:27580511

  13. Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

    PubMed Central

    Chin, Elizabeth; Kirker, Kelly; Zuck, Meghan; James, Garth; Hybiske, Kevin

    2012-01-01

    The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism. PMID

  14. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  15. GAS2 and GAS4, a Pair of Developmentally Regulated Genes Required for Spore Wall Assembly in Saccharomyces cerevisiae▿

    PubMed Central

    Ragni, Enrico; Coluccio, Alison; Rolli, Eleonora; Rodriguez-Peña, José Manuel; Colasante, Gaia; Arroyo, Javier; Neiman, Aaron M.; Popolo, Laura

    2007-01-01

    The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a β(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral. PMID:17189486

  16. Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase*

    PubMed Central

    Carroll, Sarah M.; Hampton, Randolph Y.

    2010-01-01

    Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD. PMID:19940128

  17. Federal environmental and occupational toxicology regulations and reporting requirements: a practical approach to what the medical toxicologist needs to know, part 2.

    PubMed

    Schwartz, Michael D; Dell'Aglio, Damon M; Nickle, Richard; Hornsby-Myers, Jennifer

    2014-12-01

    Toxicologists are often called upon to assist in environmental, industrial, occupational and public health assessments. Accordingly, medical toxicologists may find it prudent to be aware of applicable federal toxicological regulations and reporting requirements and of the roles of relevant federal agencies. These regulations are numerous, complex, and have evolved and expanded over time, making it difficult for toxicologists to sustain a current knowledge base. This article reviews the pertinent federal toxicological reporting requirements with regards to the Toxic Substances Control Act (TSCA), the Atomic Energy Act (AEA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Resource Conservation and Recovery Act (RCRA), the Clean Air Act, the Clean Water Act, the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), the Emergency Planning and Community Right to Know Act (EPCRA), the Occupational Safety and Health Act, the Department of Transportation, and information about the National Response Center. We reference internet-based government resources and offer direct links to applicable websites in an attempt to offer rapid and current sources of practical information. The format of the article is a series of hypothetical scenarios followed by commentary. Discussions of the Safe Drinking Water Act and the Food, Drug, and Cosmetic Act and the Dietary Supplement Health and Education Act are beyond the scope of this paper. For those desiring a more in depth discussion of the relevant federal environmental laws and statutes, and applicable case law, the reader is directed to resources such as the Environmental Law Handbook, the websites of individual laws found at www.epa.gov and the decisions of individual courts of appeal. It is our hope that this article provides not only useful practical information for the practicing toxicologist, but also serves as a key reference for Medical Toxicology core content on environmental

  18. Federal environmental and occupational toxicology regulations and reporting requirements: a practical approach to what the medical toxicologist needs to know, part 1.

    PubMed

    Schwartz, Michael D; Dell'Aglio, Damon M; Nickle, Richard; Hornsby-Myers, Jennifer

    2014-09-01

    Toxicologists are often called upon to assist in environmental, industrial, occupational and public health assessments. Accordingly, medical toxicologists may find it prudent to be aware of applicable federal toxicological regulations and reporting requirements and of the roles of relevant federal agencies. These regulations are numerous, complex, and have evolved and expanded over time, making it difficult for toxicologists to sustain a current knowledge base. This article reviews the pertinent federal toxicological reporting requirements with regard to the Toxic Substances Control Act (TSCA), the Atomic Energy Act (AEA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Resource Conservation and Recovery Act (RCRA), the Clean Air Act, the Clean Water Act, the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), the Emergency Planning and Community Right to Know Act (EPCRA), the Occupational Safety and Health Act, the Department of Transportation, and information about the National Response Center. We reference internet-based government resources and offer direct links to applicable websites in an attempt to offer rapid and current sources of practical information. The format of the article is a series of hypothetical scenarios followed by commentary. Discussions of the Safe Drinking Water Act, the Food, Drug, and Cosmetic Act, and the Dietary Supplement Health and Education Act are beyond the scope of this paper. For those desiring a more in-depth discussion of the relevant federal environmental laws and statutes and applicable case law, the reader is directed to resources such as the Environmental Law Handbook, the websites of individual laws found at www.epa.gov and the decisions of individual courts of appeal. It is our hope that this article provides not only useful practical information for the practicing toxicologist but also serves as a key reference for medical toxicology core content on environmental laws and

  19. Down-regulation of Retinoic Acid Receptor α Signaling Is Required for Sacculation and Type I Cell Formation in the Developing Lung*

    PubMed Central

    Wongtrakool, Cherry; Malpel, Sarah; Gorenstein, Julie; Sedita, Jeff; Ramirez, Maria I.; Underhill, T. Michael; Cardoso, Wellington V.

    2007-01-01

    Although retinoic acid (RA) has been shown to be critical for lung development, little is known about when RA is required and the role of individual RA receptors (RAR) in this process. Previously reported data from an RA responsive element RARE-lacZ reporter mouse show that when epithelial tubules are branching and differentiating RA signaling becomes markedly down-regulated in the epithelium. It is unclear why this down-regulation occurs and what role it might play in the developing lung. Here we analyze the effects of preventing potential progenitors of the distal lung from turning off RA signaling by locally expressing constitutively activated RARα or RARβ chimeric receptors (RARVP16) in branching airways of transgenic mice. Continued RA activation resulted in lung immaturity in both cases, but the phenotypes were remarkably different. RARαVP16 lungs did not expand to form saccules or morphologically identifiable type I cells. High levels of surfactant protein C (Sp-C), thyroid transcription factor-1 (Ttf1), and Gata6, but not Sp-A or Sp-B in the epithelium at birth suggested that in these lungs differentiation was arrested at an early stage. These alterations were not observed in RARβVP16 lungs, which showed relatively less severe changes. Our data suggest a model in which activation of RAR signaling at the onset of lung development establishes an initial program that assigns distal cell fate to the prospective lung epithelium. Down-regulation of RA signaling, however, is required to allow completion of later steps of this differentiation program that ultimately form mature type I and II cells. PMID:12947094

  20. CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity

    PubMed Central

    Monterisi, Stefania; Favia, Maria; Guerra, Lorenzo; Cardone, Rosa A.; Marzulli, Domenico; Reshkin, Stephan J.; Casavola, Valeria; Zaccolo, Manuela

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na+/H+ exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1–ezrin–actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity. PMID:22302988

  1. [Protection against electromagnetic fields 0-300 GHz in Poland. New regulations and perspectives if their harmonization wit the European Union requirements].

    PubMed

    Trzaska, Hubert

    2003-01-01

    A critical review of protection regulations (or their proposals) on work safety regarding electromagnetic fields as well as the general public protection against unwanted exposure to the fields are presented in this paper. The standards of the Western World are illogical in both aspects, biomedical knowledge and technical. The results (permissible exposures) are defined more precisely than the input data. In Poland, two independent acts are in force: one on occupational exposures and the other on non-occupational exposures. The former is "a world-better", but requires to be immediately changed to a standard that is applicable and friendly to its users. The latter can be accepted after introducing several amendments. The enlargement of the environmental standard to include occupational issues may create a good Polish proposal also useful at the international level. PMID:12924005

  2. Proper Regulation of Cdc42 Activity is Required for Tight Actin Concentration at the Equator during Cytokinesis in Adherent Mammalian Cells

    PubMed Central

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2012-01-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells. PMID:21763307

  3. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

    PubMed

    Ficarra, Florencia A; Garofalo, Cecilia G; Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction. PMID:25961560

  4. ERRγ Is Not Required for Skeletal Development but Is a RUNX2-Dependent Negative Regulator of Postnatal Bone Formation in Male Mice

    PubMed Central

    Cardelli, Marco; Aubin, Jane E.

    2014-01-01

    To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/− males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/− versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/− but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner. PMID:25313644

  5. ERRγ is not required for skeletal development but is a RUNX2-dependent negative regulator of postnatal bone formation in male mice.

    PubMed

    Cardelli, Marco; Aubin, Jane E

    2014-01-01

    To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/- males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/- versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/- but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner. PMID:25313644

  6. The J-protein AtDjB1 is required for mitochondrial complex I activity and regulates growth and development through ROS-mediated auxin signalling.

    PubMed

    Jia, Ning; Lv, Ting-Ting; Li, Mi-Xin; Wei, Shan-Shan; Li, Yan-Yi; Zhao, Chun-Lan; Li, Bing

    2016-05-01

    AtDjB1 is a mitochondria-located J-protein in Arabidopsis thaliana It is involved in the regulation of plant growth and development; however, the exact mechanisms remain to be determined. We performed comparison analyses of phenotypes, auxin signalling, redox status, mitochondrial structure and function using wild-type plants, AtDjB1 mutants, rescued AtDjB1 mutants by AtDjB1 or YUCCA2 (an auxin synthesis gene), and AtDjB1 overexpression plants. AtDjB1 mutants (atj1-1 or atj1-4) exhibited inhibition of growth and development and reductions in the level of IAA and the expression of YUCCA genes compared to wild-type plants. The introduction of AtDjB1 or YUCCA2 into atj1-1 largely rescued phenotypic defects and the IAA level, indicating that AtDjB1 probably regulates growth and development via auxin. Furthermore, atj1-1 plants displayed a significant reduction in amount/activity of mitochondrial complex I compared to wild-type plants; this resulted in the accumulation of reactive oxygen species (ROS). Moreover, exogenous H2O2 markedly inhibited the expression of YUCCA genes in wild-type plants. In contrast, the reducing agent ascorbate increased the expression of YUCCA genes and IAA level in atj1-1 plants, indicating that the low auxin level observed in atj1-1 was probably due to the high oxidation status. Overall, the data presented here suggest that AtDjB1 is required for mitochondrial complex I activity and regulates growth and development through ROS-mediated auxin signalling in Arabidopsis. PMID:27117341

  7. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

    PubMed

    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways. PMID:26872974

  8. The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence

    PubMed Central

    Gottig, Natalia; Ottado, Jorgelina

    2015-01-01

    Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family’s response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction. PMID:25961560

  9. The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

    PubMed

    Tu, Chia-Ling; Chang, Wenhan; Bikle, Daniel D

    2011-05-01

    Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation. PMID:21209619

  10. Sgs1's roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6

    PubMed Central

    Bermúdez-López, Marcelino; Villoria, María Teresa; Esteras, Miguel; Jarmuz, Adam; Torres-Rosell, Jordi; Clemente-Blanco, Andres; Aragon, Luis

    2016-01-01

    The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, from end resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1–Top3–Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1's recombination functions. PMID:27298337

  11. Glioma invasion mediated by the p75 neurotrophin receptor (p75NTR/CD271) requires regulated interaction with PDLIM1

    PubMed Central

    Ahn, B Y; Saldanha-Gama, R F G; Rahn, J J; Hao, X; Zhang, J; Dang, N-H; Alshehri, M; Robbins, S M; Senger, D L

    2016-01-01

    The invasive nature of glioblastoma renders them incurable by current therapeutic interventions. Using a novel invasive human glioma model, we previously identified the neurotrophin receptor p75NTR (aka CD271) as a mediator of glioma invasion. Herein, we provide evidence that preventing phosphorylation of p75NTR on S303 by pharmacological inhibition of PKA, or by a mutational strategy (S303G), cripples p75NTR-mediated glioma invasion resulting in serine phosphorylation within the C-terminal PDZ-binding motif (SPV) of p75NTR. Consistent with this, deletion (ΔSPV) or mutation (SPM) of the PDZ motif results in abrogation of p75NTR-mediated invasion. Using a peptide-based strategy, we identified PDLIM1 as a novel signaling adaptor for p75NTR and provide the first evidence for a regulated interaction via S425 phosphorylation. Importantly, PDLIM1 was shown to interact with p75NTR in highly invasive patient-derived glioma stem cells/tumor-initiating cells and shRNA knockdown of PDLIM1 in vitro and in vivo results in complete ablation of p75NTR-mediated invasion. Collectively, these data demonstrate a requirement for a regulated interaction of p75NTR with PDLIM1 and suggest that targeting either the PDZ domain interactions and/or the phosphorylation of p75NTR by PKA could provide therapeutic strategies for patients with glioblastoma. PMID:26119933

  12. Requirement of miR-9-dependent regulation of Myocd in PASMCs phenotypic modulation and proliferation induced by hepatopulmonary syndrome rat serum

    PubMed Central

    Xu, Duo; Gu, Jian-teng; Yi, Bin; Chen, Lin; Wang, Guan-song; Qian, Gui-sheng; Lu, Kai-zhi

    2015-01-01

    Hepatopulmonary syndrome (HPS) is characterized by a triad of severe liver disease, intrapulmonary vascular dilation and hypoxaemia. Pulmonary vascular remodelling (PVR) is a key feature of HPS pathology. Our previous studies have established the role of the pulmonary artery smooth muscle cell (PASMC) phenotypic modulation and proliferation in HPS-associated PVR. Myocardin, a robust transcriptional coactivator of serum response factor, plays a critical role in the vascular smooth muscle cell phenotypic switch. However, the mechanism regulating myocardin upstream signalling remains unclear. In this study, treatment of rat PASMCs with serum drawn from common bile duct ligation rats, which model symptoms of HPS, resulted in a significant increase in miR-9 expression correlated with a decrease in expression of myocardin and the phenotypic markers SM-α-actin and smooth muscle-specific myosin heavy chain (SM-MHC). Furthermore, miRNA functional analysis and luciferase reporter assay demonstrated that miR-9 effectively regulated myocardin expression by directly binding to its 3′-untranslated region. Both the knockdown of miR-9 and overexpression of myocardin effectively attenuated the HPS rat serum-induced phenotype switch and proliferation of PASMCs. Taken together, the findings of our present study demonstrate that miR-9 is required in HPS rat serum-induced phenotypic modulation and proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential therapeutic target in HPS. PMID:26147104

  13. Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by hypoxia-inducible factor 2α

    PubMed Central

    Lien, Huang-Wei; Yuan, Rey-Yue; Chou, Chih-Ming; Chen, Yi-Chung; Hung, Chin-Chun; Hu, Chin-Hwa; Hwang, Sheng-Ping L.; Hwang, Pung-Pung; Shen, Chia-Ning; Chen, Chih-Lung; Cheng, Chia-Hsiung; Huang, Chang-Jen

    2016-01-01

    Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons. PMID:27323909

  14. Post-prandial regulation of hepatic glucokinase and lipogenesis requires the activation of TORC1 signalling in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Dai, Weiwei; Panserat, Stéphane; Mennigen, Jan A; Terrier, Frédéric; Dias, Karine; Seiliez, Iban; Skiba-Cassy, Sandrine

    2013-12-01

    To assess the potential involvement of TORC1 (target of rapamycin complex 1) signalling in the regulation of post-prandial hepatic lipid and glucose metabolism-related gene expression in trout, we employed intraperitoneal administration of rapamycin to achieve an acute inhibition of the TOR pathway. Our results reveal that rapamycin inhibits the phosphorylation of TORC1 and its downstream effectors (S6K1, S6 and 4E-BP1), without affecting Akt and the Akt substrates Forkhead-box Class O1 (FoxO1) and glycogen synthase kinase 3α/β (GSK 3α/β). These results indicate that acute administration of rapamycin in trout leads to the inhibition of TORC1 activation. No effect is observed on the expression of genes involved in gluconeogenesis, glycolysis and fatty acid oxidation, but hepatic TORC1 inhibition results in decreased sterol regulatory element binding protein 1c (SREBP1c) gene expression and suppressed fatty acid synthase (FAS) and glucokinase (GK) at gene expression and activity levels, indicating that FAS and GK activity is controlled at a transcriptional level in a TORC1-dependent manner. This study demonstrates for the first time in fish that post-prandial regulation of hepatic lipogenesis and glucokinase in rainbow trout requires the activation of TORC1 signalling. PMID:24031053

  15. Negative Feedback Regulation of the Yeast Cth1 and Cth2 mRNA Binding Proteins Is Required for Adaptation to Iron Deficiency and Iron Supplementation

    PubMed Central

    Martínez-Pastor, Mar; Vergara, Sandra V.

    2013-01-01

    Iron (Fe) is an essential element for all eukaryotic organisms because it functions as a cofactor in a wide range of biochemical processes. Cells have developed sophisticated mechanisms to tightly control Fe utilization in response to alterations in cellular demands and bioavailability. In response to Fe deficiency, the yeast Saccharomyces cerevisiae activates transcription of the CTH1 and CTH2 genes, which encode proteins that bind to AU-rich elements (AREs) within the 3′ untranslated regions (3′UTRs) of many mRNAs, leading to metabolic reprogramming of Fe-dependent pathways and decreased Fe storage. The precise mechanisms underlying Cth1 and Cth2 function and regulation are incompletely understood. We report here that the Cth1 and Cth2 proteins specifically bind in vivo to AREs located at the 3′UTRs of their own transcripts in an auto- and cross-regulated mechanism that limits their expression. By mutagenesis of the AREs within the CTH2 transcript, we demonstrate that a Cth2 negative-feedback loop is required for the efficient decline in Cth2 protein levels observed upon a rapid rise in Fe availability. Importantly, Cth2 autoregulation is critical for the appropriate recovery of Fe-dependent processes and resumption of growth in response to a change from Fe deficiency to Fe supplementation. PMID:23530061

  16. crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis.

    PubMed

    D'Avino, P P; Thummel, C S

    1998-05-01

    Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis. PMID:9521911

  17. Requirement of miR-9-dependent regulation of Myocd in PASMCs phenotypic modulation and proliferation induced by hepatopulmonary syndrome rat serum.

    PubMed

    Xu, Duo; Gu, Jian-teng; Yi, Bin; Chen, Lin; Wang, Guan-song; Qian, Gui-sheng; Lu, Kai-zhi

    2015-10-01

    Hepatopulmonary syndrome (HPS) is characterized by a triad of severe liver disease, intrapulmonary vascular dilation and hypoxaemia. Pulmonary vascular remodelling (PVR) is a key feature of HPS pathology. Our previous studies have established the role of the pulmonary artery smooth muscle cell (PASMC) phenotypic modulation and proliferation in HPS-associated PVR. Myocardin, a robust transcriptional coactivator of serum response factor, plays a critical role in the vascular smooth muscle cell phenotypic switch. However, the mechanism regulating myocardin upstream signalling remains unclear. In this study, treatment of rat PASMCs with serum drawn from common bile duct ligation rats, which model symptoms of HPS, resulted in a significant increase in miR-9 expression correlated with a decrease in expression of myocardin and the phenotypic markers SM-α-actin and smooth muscle-specific myosin heavy chain (SM-MHC). Furthermore, miRNA functional analysis and luciferase reporter assay demonstrated that miR-9 effectively regulated myocardin expression by directly binding to its 3'-untranslated region. Both the knockdown of miR-9 and overexpression of myocardin effectively attenuated the HPS rat serum-induced phenotype switch and proliferation of PASMCs. Taken together, the findings of our present study demonstrate that miR-9 is required in HPS rat serum-induced phenotypic modulation and proliferation of PASMCs for targeting of myocardin and that miR-9 may serve as a potential therapeutic target in HPS. PMID:26147104

  18. The master regulator for biofilm formation in Bacillus subtilis governs the expression of an operon encoding secreted proteins required for the assembly of complex multicellular communities.

    SciTech Connect

    Branda, Steven S.; Losick, Richard; Kolter, Roberto; Kearns, Daniel B.; Chu, Frances

    2005-08-01

    Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR-mediated repression. We further propose that SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.

  19. Glioma invasion mediated by the p75 neurotrophin receptor (p75(NTR)/CD271) requires regulated interaction with PDLIM1.

    PubMed

    Ahn, B Y; Saldanha-Gama, R F G; Rahn, J J; Hao, X; Zhang, J; Dang, N-H; Alshehri, M; Robbins, S M; Senger, D L

    2016-03-17

    The invasive nature of glioblastoma renders them incurable by current therapeutic interventions. Using a novel invasive human glioma model, we previously identified the neurotrophin receptor p75(NTR) (aka CD271) as a mediator of glioma invasion. Herein, we provide evidence that preventing phosphorylation of p75(NTR) on S303 by pharmacological inhibition of PKA, or by a mutational strategy (S303G), cripples p75(NTR)-mediated glioma invasion resulting in serine phosphorylation within the C-terminal PDZ-binding motif (SPV) of p75(NTR). Consistent with this, deletion (ΔSPV) or mutation (SPM) of the PDZ motif results in abrogation of p75(NTR)-mediated invasion. Using a peptide-based strategy, we identified PDLIM1 as a novel signaling adaptor for p75(NTR) and provide the first evidence for a regulated interaction via S425 phosphorylation. Importantly, PDLIM1 was shown to interact with p75(NTR) in highly invasive patient-derived glioma stem cells/tumor-initiating cells and shRNA knockdown of PDLIM1 in vitro and in vivo results in complete ablation of p75(NTR)-mediated invasion. Collectively, these data demonstrate a requirement for a regulated interaction of p75(NTR) with PDLIM1 and suggest that targeting either the PDZ domain interactions and/or the phosphorylation of p75(NTR) by PKA could provide therapeutic strategies for patients with glioblastoma. PMID:26119933

  20. The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner

    PubMed Central

    Richmond, Grace E.; Evans, Laura P.; Anderson, Michele J.; Wand, Matthew E.; Bonney, Laura C.; Ivens, Alasdair; Chua, Kim Lee; Webber, Mark A.; Sutton, J. Mark; Peterson, Marnie L.

    2016-01-01

    ABSTRACT The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery. PMID:27094331

  1. Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems.

    PubMed

    Buzeli, Reginaldo A A; Cascardo, Júlio C M; Rodrigues, Leonardo A Z; Andrade, Maxuel O; Almeida, Raul S; Loureiro, Marcelo E; Otoni, Wagner C; Fontes, Elizabeth P B

    2002-11-01

    The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5' flanking sequences were fused to beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (-358 to -211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (-211 to -80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter. PMID:12374306

  2. Tomato LeTHIC is an Fe-requiring HMP-P synthase involved in thiamine synthesis and regulated by multiple factors.

    PubMed

    Zhao, Weina; Cheng, Xudong; Huang, Zongan; Fan, Huajie; Wu, Huilan; Ling, Hong-Qing

    2011-06-01

    Thiamine is a key primary metabolite which is necessary for the viability of all organisms. It is a dietary requirement for mammals because only prokaryotes, fungi and plants are thiamine prototrophs. In contrast to the well documented biosynthetic mechanism in bacteria, much remains to be deciphered in plants. In this work, a tomato thiamine-auxotrophic (thiamineless, tl) mutant was characterized. The tl mutant occurs due to inactivation of LeTHIC transcription as a result of insertion of a large unknown DNA fragment in its 5'-untranslated region. Expression of wild-type LeTHIC in tl plants was able to complement the mutant to wild type. LeTHIC possessed the same function as E.cTHIC [an Escherichia coli 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase involved in synthesis of the pyrimidine moiety of thiamine] because expression of LeTHIC rescued THIC-deficient strains of E. coli under culture conditions without thiamine supplementation, suggesting that plants employ a bacteria-like route of pyrimidine moiety synthesis. LeTHIC is an Fe-S cluster protein localized in chloroplasts, and Fe is required for maintenance of its enzyme activity because Fe deficiency resulted in a significant reduction of thiamine content in tomato leaves. Further, we also showed that the expression of LeTHIC is tightly regulated at the transcriptional and post-transcriptional level by multiple factors, such as light, Fe status and thiamine pyrophosphate (TPP)-riboswitch. The results clearly demonstrated that a feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato. Our results provide a new insight into understanding the molecular mechanism of thiamine biosynthesis in plants. PMID:21511719

  3. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

    PubMed

    Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

    2013-08-28

    Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads. PMID:23986233

  4. Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2.

    PubMed

    Sowa, Hideaki; Kaji, Hiroshi; Hendy, Geoffrey N; Canaff, Lucie; Komori, Toshihisa; Sugimoto, Toshitsugu; Chihara, Kazuo

    2004-09-24

    Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late

  5. Vehicle Efficiency and Tractive Work: Rate of Change for the Past Decade and Accelerated Progress Required for U.S. Fuel Economy and CO2 Regulations

    DOE PAGESBeta

    Thomas, John

    2016-04-05

    A major driving force for change in light-duty vehicle design and technology is the National Highway Traffic Safety Administration (NHTSA) and the U.S. Environmental Protection Agency (EPA) joint final rules concerning Corporate Average Fuel Economy (CAFE) and greenhouse gas (GHG) emissions for model years (MY) 2016 through 2025 passenger cars and light trucks. The chief goal of this current study is to compare the already rapid pace of fuel economy improvement and technological change over the previous decade to the needed rate of change to meet regulations over the next decade. EPA and NHTSA comparisons of the MY 2004 USmore » light-duty vehicle fleet to the MY 2014 fleet shows improved fuel economy (FE) of approximately 28% using the same FE estimating method mandated for CAFE regulations. Future predictions by EPA and NHTSA concerning ensemble fleet fuel economy are examined as an indicator of needed vehicle rate-of-change. A set of 40 same-model vehicle pairs for MY 2005 and MY 2015 is compared to examine changes in energy use and related technological change over the 10 year period. Powertrain improvements measured as increased vehicle efficiency, and vehicle mass-glider improvements measured as decreased tractive work requirements are quantified. The focus is first on conventional gasoline powertrain vehicles which currently dominate the market, with hybrids also examined due to their high potential importance for CAFE compliance. Most hybrid vehicles with significant sales in 2014 were represented in the study. Results show 10 years of progress for the studied vehicle set includes lowered tractive effort of about 5.6% and improved powertrain efficiency of about 16.5%. Further analysis shows that this high rate of past progress must increase by about 50% in order to meet the 2025 CAFE standards. Examination of where certain MY 2015 vehicle compare to CAFE regulations is offered as well as some simple conjecture on what is needed to meet regulations under

  6. The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence

    PubMed Central

    Lukassen, Susanne; Späth, Michaela; Wolferstätter, Michael; Babel, Eveline; Brinkmann, Kay; Wielert, Ursula; Chaplin, Paul; Suter, Mark

    2012-01-01

    Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo. PMID:22171261

  7. Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation.

    PubMed

    Schalm, Stefanie S; Tee, Andrew R; Blenis, John

    2005-03-25

    The mammalian target of rapamycin, mTOR, is a Ser/Thr kinase that promotes cell growth and proliferation by activating ribosomal protein S6 kinase 1 (S6K1). We previously identified a conserved TOR signaling (TOS) motif in the N terminus of S6K1 that is required for its mTOR-dependent activation. Furthermore, our data suggested that the TOS motif suppresses an inhibitory function associated with the C terminus of S6K1. Here, we have characterized the mTOR-regulated inhibitory region within the C terminus. We have identified a conserved C-terminal "RSPRR" sequence that is responsible for an mTOR-dependent suppression of S6K1 activation. Deletion or mutations within this RSPRR motif partially rescue the kinase activity of the S6K1 TOS motif mutant (S6K1-F5A), and this rescued activity is rapamycin resistant. Furthermore, we have shown that the RSPRR motif significantly suppresses S6K1 phosphorylation at two phosphorylation sites (Thr-389 and Thr-229) that are crucial for S6K1 activation. Importantly, introducing both the Thr-389 phosphomimetic and RSPRR motif mutations into the catalytically inactive S6K1 mutant S6K1-F5A completely rescues its activity and renders it fully rapamycin resistant. These data show that the N-terminal TOS motif suppresses an inhibitory function mediated by the C-terminal RSPRR motif. We propose that the RSPRR motif interacts with a negative regulator of S6K1 that is normally suppressed by mTOR. PMID:15659381

  8. Preschool Regulations.

    ERIC Educational Resources Information Center

    Nebraska State Dept. of Health and Human Services, Lincoln.

    Published by the Department of Health and Human Services, as required by Nebraska law, this guide details regulations for the physical well-being, safety, and protection of children and defines the minimum levels of acceptable services to be provided in Nebraska preschools. The first section of the guide lists specific preschool regulations,…

  9. 14 CFR 11.61 - May I ask FAA to adopt, amend, or repeal a regulation, or grant relief from the requirements of a...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Code of Federal Regulations (14 CFR) or ask FAA to amend or repeal a current regulation in 14 CFR. (b) Using a petition for exemption, you may ask FAA to grant you relief from current regulations in 14 CFR. ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false May I ask FAA to adopt, amend, or repeal...

  10. The dicer-like1 Homolog fuzzy tassel Is Required for the Regulation of Meristem Determinacy in the Inflorescence and Vegetative Growth in Maize[W][OPEN

    PubMed Central

    Thompson, Beth E.; Basham, Christine; Hammond, Reza; Ding, Queying; Kakrana, Atul; Lee, Tzuu-Fen; Simon, Stacey A.; Meeley, Robert; Hake, Sarah

    2014-01-01

    Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles. PMID:25465405

  11. Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2.

    PubMed

    Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José Manuel; Nombela, César; Arroyo, Javier

    2016-09-01

    The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway. PMID:27112564

  12. Iron-regulated metabolites produced by Pseudomonas fluorescens WCS374r are not required for eliciting induced systemic resistance against Pseudomonas syringae pv. tomato in Arabidopsis

    PubMed Central

    Djavaheri, Mohammad; Mercado-Blanco, Jesús; Versluis, C; Meyer, J-M; Loon, L C; Bakker, Peter A H M

    2012-01-01

    The plant growth-promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron-regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere-colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis. PMID:23170230

  13. Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA

    PubMed Central

    Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

    2015-01-01

    Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1–HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. PMID:25628358

  14. A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling, thereby reducing energy requirements during ischemia.

    PubMed

    Wang, L-M; Wang, Y-J; Cui, M; Luo, W-J; Wang, X-J; Barber, P A; Chen, Z-Y

    2016-04-01

    The above article from European Journal of Neuroscience, published online on 5 March 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1111/ejn.12162/full), has been retracted by agreement between the Editors-in-Chief, Paul Bolam and John Foxe, the authors and John Wiley & Sons Ltd. The retraction has been agreed as Dr Phillip Barber has informed the publisher that he had seen neither the original data nor any version of the manuscript, and had not been involved in the work reported. A subsequent Institutional investigation found evidence of misconduct on the part of the submitting author. Reference Wang, L.-M., Wang, Y.-J., Cui, M., Luo, W.-J., Wang, X.-J., Barber, P.A. & Chen, Z.-Y. (2013) A dietary polyphenol resveratrol acts to provide neuroprotection in recurrent stroke models by regulating AMPK and SIRT1 signaling, thereby reducing energy requirements during ischemia. Eur. J. Neurosci., 37, 1669-1681. doi: 10.1111/ejn.12162. PMID:27041235

  15. Regulation of Tlx3 by Pax6 is required for the restricted expression of Chrnα3 in Cerebellar Granule Neuron progenitors during development

    PubMed Central

    Divya, Thulasi Sheela; Lalitha, Soundararajan; Parvathy, Surendran; Subashini, Chandramohan; Sanalkumar, Rajendran; Dhanesh, Sivadasan Bindu; Rasheed, Vazhanthodi Abdul; Divya, Mundackal Sivaraman; Tole, Shubha; James, Jackson

    2016-01-01

    Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6−/−Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD). PMID:27452274

  16. Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2

    PubMed Central

    Sanz, Ana Belén; García, Raúl; Rodríguez-Peña, José Manuel; Nombela, César; Arroyo, Javier

    2016-01-01

    The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway. PMID:27112564

  17. Checkpoint kinase 1 (Chk1) is required for mitotic progression through negative regulation of polo-like kinase 1 (Plk1)

    PubMed Central

    Tang, Jiabin; Erikson, Raymond L.; Liu, Xiaoqi

    2006-01-01

    Although the essential function of checkpoint kinase 1 (Chk1) in DNA damage response has been well established, the role of Chk1 in normal cell cycle progression is unclear. By using RNAi to specifically deplete Chk1, we determined loss-of-function phenotypes in HeLa cells. A vector-based RNAi approach showed that Chk1 is required for normal cell proliferation and survival, inasmuch as a dramatic cell-cycle arrest at G2/M phase and massive apoptosis were observed in Chk1-deficient cells. Coupling of siRNA with cell synchronization further revealed that Chk1 depletion leads to metaphase block, as indicated by various mitotic markers. Neither bipolar spindle formation nor centrosome functions were affected by Chk1 depletion; however, the depleted cells exhibited chromosome misalignment during metaphase, chromosome lagging during anaphase, and kinetochore defects within the regions of misaligned/lagging chromosomes. Moreover, we showed that Chk1 is a negative regulator of polo-like kinase 1 (Plk1), in either the absence or presence of DNA damage. Finally, Chk1 depletion leads to the activation of the spindle checkpoint because codepletion of spindle checkpoint proteins rescues the Chk1 depletion-induced mitotic arrest. PMID:16873548

  18. Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

    PubMed Central

    Seaberg, Bonnie; Henslee, Gabrielle; Wang, Shuo; Paez-Colasante, Ximena; Landreth, Gary E.

    2015-01-01

    The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice. PMID:25605336

  19. The dicer-like1 homolog fuzzy tassel is required for the regulation of meristem determinacy in the inflorescence and vegetative growth in maize.

    PubMed

    Thompson, Beth E; Basham, Christine; Hammond, Reza; Ding, Queying; Kakrana, Atul; Lee, Tzuu-Fen; Simon, Stacey A; Meeley, Robert; Meyers, Blake C; Hake, Sarah

    2014-12-01

    Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles. PMID:25465405

  20. Extracellular Signal-regulated Kinase Activation Is Required for Serine 727 Phosphorylation of STAT3 in Schwann Cells in vitro and in vivo

    PubMed Central

    Lee, Hyun Kyoung; Jung, Junyang; Lee, Sang Hwa; Seo, Su-Yeong; Suh, Duk Joon

    2009-01-01

    In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury. PMID:19885032

  1. Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2.

    PubMed

    Robledo, Marta; Jiménez-Zurdo, José I; Soto, M José; Velázquez, Encarnación; Dazzo, Frank; Martínez-Molina, Eustoquio; Mateos, Pedro F

    2011-07-01

    The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis. PMID:21405987

  2. Vernalization Requirement and the Chromosomal VRN1-Region can Affect Freezing Tolerance and Expression of Cold-Regulated Genes in Festuca pratensis

    PubMed Central

    Ergon, Åshild; Melby, Tone I.; Höglind, Mats; Rognli, Odd A.

    2016-01-01

    Plants adapted to cold winters go through annual cycles of gain followed by loss of freezing tolerance (cold acclimation and deacclimation). Warm spells during winter and early spring can cause deacclimation, and if temperatures drop, freezing damage may occur. Many plants are vernalized during winter, a process making them competent to flower in the following summer. In winter cereals, a coincidence in the timing of vernalization saturation, deacclimation, downregulation of cold-induced genes, and reduced ability to reacclimate, occurs under long photoperiods and is under control of the main regulator of vernalization requirement in cereals, VRN1, and/or closely linked gene(s). Thus, the probability of freezing damage after a warm spell may depend on both vernalization saturation and photoperiod. We investigated the role of vernalization and the VRN1-region on freezing tolerance of meadow fescue (Festuca pratensis Huds.), a perennial grass species. Two F2 populations, divergently selected for high and low vernalization requirement, were studied. Each genotype was characterized for the copy number of one of the four parental haplotypes of the VRN1-region. Clonal plants were cold acclimated for 2 weeks or vernalized/cold acclimated for a total of 9 weeks, after which the F2 populations reached different levels of vernalization saturation. Vernalized and cold acclimated plants were deacclimated for 1 week and then reacclimated for 2 weeks. All treatments were given at 8 h photoperiod. Flowering response, freezing tolerance and expression of the cold-induced genes VRN1, MADS3, CBF6, COR14B, CR7 (BLT14), LOS2, and IRI1 was measured. We found that some genotypes can lose some freezing tolerance after vernalization and a deacclimation–reacclimation cycle. The relationship between vernalization and freezing tolerance was complex. We found effects of the VRN1-region on freezing tolerance in plants cold acclimated for 2 weeks, timing of heading after 9 weeks of

  3. Vernalization Requirement and the Chromosomal VRN1-Region can Affect Freezing Tolerance and Expression of Cold-Regulated Genes in Festuca pratensis.

    PubMed

    Ergon, Åshild; Melby, Tone I; Höglind, Mats; Rognli, Odd A

    2016-01-01

    Plants adapted to cold winters go through annual cycles of gain followed by loss of freezing tolerance (cold acclimation and deacclimation). Warm spells during winter and early spring can cause deacclimation, and if temperatures drop, freezing damage may occur. Many plants are vernalized during winter, a process making them competent to flower in the following summer. In winter cereals, a coincidence in the timing of vernalization saturation, deacclimation, downregulation of cold-induced genes, and reduced ability to reacclimate, occurs under long photoperiods and is under control of the main regulator of vernalization requirement in cereals, VRN1, and/or closely linked gene(s). Thus, the probability of freezing damage after a warm spell may depend on both vernalization saturation and photoperiod. We investigated the role of vernalization and the VRN1-region on freezing tolerance of meadow fescue (Festuca pratensis Huds.), a perennial grass species. Two F2 populations, divergently selected for high and low vernalization requirement, were studied. Each genotype was characterized for the copy number of one of the four parental haplotypes of the VRN1-region. Clonal plants were cold acclimated for 2 weeks or vernalized/cold acclimated for a total of 9 weeks, after which the F2 populations reached different levels of vernalization saturation. Vernalized and cold acclimated plants were deacclimated for 1 week and then reacclimated for 2 weeks. All treatments were given at 8 h photoperiod. Flowering response, freezing tolerance and expression of the cold-induced genes VRN1, MADS3, CBF6, COR14B, CR7 (BLT14), LOS2, and IRI1 was measured. We found that some genotypes can lose some freezing tolerance after vernalization and a deacclimation-reacclimation cycle. The relationship between vernalization and freezing tolerance was complex. We found effects of the VRN1-region on freezing tolerance in plants cold acclimated for 2 weeks, timing of heading after 9 weeks of

  4. Induction of Proinflammatory Responses in Macrophages by the Glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum: CELL SIGNALING RECEPTORS, GPI STRUCTURAL REQUIREMENT, AND REGULATION OF GPI ACTIVITY*

    PubMed Central

    Krishnegowda, Gowdahalli; Hajjar, Adeline M.; Zhu, Jianzhong; Douglass, Erika J.; Uematsu, Satoshi; Akira, Shizuo; Woods, Amina S.; Gowda, D. Channe

    2016-01-01

    SUMMARY The proinflammatory cytokines produced by the innate immune system in response to pathogenic infection protect the host by controlling microbial growth. However, excessive proinflammatory responses could disrupt the host’s vital physiological functions, causing severe pathological conditions. In the case of Plasmodium falciparum, the protozoan parasite that causes fatal malaria in man, the glycosylphosphatidylinositol (GPI) anchors are thought to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study, we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses, and studied the GPI structure-activity relationship. The data show that GPI-signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2 lyso GPIs is comparable to that of the intact GPIs, whereas the activity of Man3-GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man3-GPIs) and two fatty acids (sn-2 lyso GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of ERK, JNK and p38, and NF-κB signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., et al. (2004) J. Biol. Chem., accompanying manuscript). Our data also show that GPIs are degraded by the macrophage surface phospholipases, predominantly into inactive species, indicating that the host can regulate GPI activity, at least in part, by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate

  5. The Pal pathway required for ambient pH adaptation regulates growth, conidiation, and osmotolerance of Beauveria bassiana in a pH-dependent manner.

    PubMed

    Zhu, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2016-05-01

    The Pal/Rim pathway essential for fungal adaptation to ambient pH has been unexplored in Beauveria bassiana, a classic fungal entomopathogen. Here, we show the characterized Pal pathway comprising transcription factor PacC and upstream six Pal partners (PalA/B/C/F/H/I) in B. bassiana. Their coding genes were all transcribed most abundantly in standard wild-type culture under the alkaline condition of pH 9. Deletion of pacC or each pal gene resulted in a significant delay of culture acidification in a minimal broth (initial pH = 7.3). This delay concurred with altered accumulation levels of intra/extracellular organic acids and drastically depressed expression of some enzyme genes required for the syntheses of oxalic and lactic acids. Our deletion mutants except ΔpalI showed growth defects and maximal sensitivity to NaCl, KCl, LiCl, or sorbitol at pH 9, an alkaline condition leading to fragmented vacuoles in their hyphal cells exposed to osmotic stress. In these mutants, conidiation was significantly facilitated at pH 3 more than at pH 7 but suppressed slightly at pH 9. Mild virulence defects also occurred in the absence of pacC or any pal gene. These changes were restored by targeted gene complementation. Taken together, PacC and Pal partners regulate the growth, conidiation, and osmotolerance of B. bassiana in a pH-dependent manner, highlighting their vitality for the fungal pH response. PMID:26754817

  6. Poly(I:C) Induces Antiviral Immune Responses in Japanese Flounder (Paralichthys olivaceus) That Require TLR3 and MDA5 and Is Negatively Regulated by Myd88

    PubMed Central

    Zhou, Zhi-xia; Zhang, Bao-cun; Sun, Li

    2014-01-01

    Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish. PMID:25393122

  7. Chronic activation of extracellular-signal-regulated protein kinases by phenylephrine is required to elicit a hypertrophic response in cardiac myocytes.

    PubMed Central

    Barron, Anthony J; Finn, Stephen G; Fuller, Stephen J

    2003-01-01

    Extracellular-signal-regulated protein kinases (ERKs) are activated rapidly and transiently in response to phenylephrine (PE) and endothelin-1 (ET-1) in cardiac myocytes, but whether this is linked to the subsequent development of the hypertrophic phenotype remains equivocal. To investigate this, we examined the dependence of the hypertrophic response on the length of exposure to PE in neonatal myocyte cultures. In addition to the initial transient activation of ERKs (maximum at 5-10 min), PE (10 microM) induced a second, more prolonged peak of activity several hours later. The activity of a transfected atrial natriuretic factor-luciferase reporter gene was increased 10- to 24-fold by PE. This response was inhibited by the alpha(1)-antagonist prazosin (100 nM) and by U0126 (10 microM) and PD184352 (1 microM), inhibitors of ERK activation, irrespective of whether these were added before or up to 24 h after the addition of PE. Prazosin had no effect on ET-1 (50 nM)-stimulated atrial natriuretic factor-luciferase activity. Protein synthesis was enhanced by 35+/-6% by PE, and this was blocked by prazosin added 1 h after the addition of PE, but decreased only by half when added 8 h after PE. Similarly, PE (48 h) increased myocyte area by 49% and this was prevented by prazosin added 1 h after PE, but decreased only by half when added at 24 h. These results demonstrate that prolonged exposure to PE is required to elicit alterations in gene expression, protein synthesis and cell size, characteristic of hypertrophied myocytes, and they confirm that the initial peak of ERK activity is insufficient to trigger hypertrophic responses. PMID:12513686

  8. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

    PubMed Central

    Remacha, M; Jimenez-Diaz, A; Bermejo, B; Rodriguez-Gabriel, M A; Guarinos, E; Ballesta, J P

    1995-01-01

    Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. PMID:7651393

  9. Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function.

    PubMed

    Waldman, Maayan; Bellner, Lars; Vanella, Luca; Schragenheim, Joseph; Sodhi, Komal; Singh, Shailendra P; Lin, Daohong; Lakhkar, Anand; Li, Jiangwei; Hochhauser, Edith; Arad, Michael; Darzynkiewicz, Zbigniew; Kappas, Atallah; Abraham, Nader G

    2016-07-15

    Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhanced peroxisome proliferation-activated receptor γ coactivator α (PGC-1α) expression, and increased mitochondrial function in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase, P < 0.05, in PGC-1α and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase in heme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1α as cells deficient in PGC-1α (PGC-1α knockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treated with EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1α-dependent manner, P < 0.05. The increase in PGC-1α was associated with an increase in β-catenin, P < 0.05, adiponectin expression, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS (MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specific transcript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/paracrine manner to increase PGC-1α is required for activation of HO-1 expression. This is the first study to dissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1α signaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1 expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargement and terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in the canonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation. PMID:27224420

  10. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo

    PubMed Central

    Eskin, Julian A.; Rankova, Aneliya; Johnston, Adam B.; Alioto, Salvatore L.; Goode, Bruce L.

    2016-01-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles. PMID:26764093

  11. Ssn6 Defines a New Level of Regulation of White-Opaque Switching in Candida albicans and Is Required For the Stochasticity of the Switch

    PubMed Central

    Lohse, Matthew B.; Nobile, Clarissa J.; Noiman, Liron; Laksana, Clement N.

    2016-01-01

    ABSTRACT The human commensal and opportunistic pathogen Candida albicans can switch between two distinct, heritable cell types, named “white” and “opaque,” which differ in morphology, mating abilities, and metabolic preferences and in their interactions with the host immune system. Previous studies revealed a highly interconnected group of transcriptional regulators that control switching between the two cell types. Here, we identify Ssn6, the C. albicans functional homolog of the Saccharomyces cerevisiae transcriptional corepressor Cyc8, as a new regulator of white-opaque switching. In a or α mating type strains, deletion of SSN6 results in mass switching from the white to the opaque cell type. Transcriptional profiling of ssn6 deletion mutant strains reveals that Ssn6 represses part of the opaque cell transcriptional program in white cells and the majority of the white cell transcriptional program in opaque cells. Genome-wide chromatin immunoprecipitation experiments demonstrate that Ssn6 is tightly integrated into the opaque cell regulatory circuit and that the positions to which it is bound across the genome strongly overlap those bound by Wor1 and Wor2, previously identified regulators of white-opaque switching. This work reveals the next layer in the white-opaque transcriptional circuitry by integrating a transcriptional regulator that does not bind DNA directly but instead associates with specific combinations of DNA-bound transcriptional regulators. PMID:26814177

  12. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo.

    PubMed

    Eskin, Julian A; Rankova, Aneliya; Johnston, Adam B; Alioto, Salvatore L; Goode, Bruce L

    2016-03-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1's functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles. PMID:26764093

  13. Up-Regulation of TREK-2 Potassium Channels in Cultured Astrocytes Requires De Novo Protein Synthesis: Relevance to Localization of TREK-2 Channels in Astrocytes after Transient Cerebral Ischemia

    PubMed Central

    Rivera-Pagán, Aixa F.; Rivera-Aponte, David E.; Melnik-Martínez, Katya V.; Zayas-Santiago, Astrid; Kucheryavykh, Lilia Y.; Martins, Antonio H.; Cubano, Luis A.; Skatchkov, Serguei N.; Eaton, Misty J.

    2015-01-01

    Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role. PMID:25886567

  14. A novel fur- and iron-regulated small RNA, NrrF, is required for indirect fur-mediated regulation of the sdhA and sdhC genes in Neisseria meningitidis.

    PubMed

    Mellin, J R; Goswami, Sulip; Grogan, Susan; Tjaden, Brian; Genco, Caroline A

    2007-05-01

    Iron is both essential for bacterial growth and toxic at higher concentrations; thus, iron homeostasis is tightly regulated. In Neisseria meningitidis the majority of iron-responsive gene regulation is mediated by the ferric uptake regulator protein (Fur), a protein classically defined as a transcriptional repressor. Recently, however, microarray studies have identified a number of genes in N. meningitidis that are iron and Fur activated, demonstrating a new role for Fur as a transcriptional activator. Since Fur has been shown to indirectly activate gene transcription through the repression of small regulatory RNA molecules in other organisms, we hypothesized that a similar mechanism could account for Fur-dependent, iron-activated gene transcription in N. meningitidis. In this study, we used a bioinformatics approach to screen for the presence of Fur-regulated small RNA molecules in N. meningitidis MC58. This screen identified one small RNA, herein named NrrF (for neisserial regulatory RNA responsive to iron [Fe]), which was demonstrated to be both iron responsive and Fur regulated and which has a well-conserved orthologue in N. gonorrhoeae. In addition, this screen identified a number of other likely, novel small RNA transcripts. Lastly, we utilized a new bioinformatics approach to predict regulatory targets of the NrrF small RNA. This analysis led to the identification of the sdhA and sdhC genes, which were subsequently demonstrated to be under NrrF regulation in an nrrF mutant. This study is the first report of small RNAs in N. meningitidis and the first to use a bioinformatics approach to identify, a priori, regulatory targets of a small RNA. PMID:17351036

  15. A Novel Fur- and Iron-Regulated Small RNA, NrrF, Is Required for Indirect Fur-Mediated Regulation of the sdhA and sdhC Genes in Neisseria meningitidis▿ †

    PubMed Central

    Mellin, J. R.; Goswami, Sulip; Grogan, Susan; Tjaden, Brian; Genco, Caroline A.

    2007-01-01

    Iron is both essential for bacterial growth and toxic at higher concentrations; thus, iron homeostasis is tightly regulated. In Neisseria meningitidis the majority of iron-responsive gene regulation is mediated by the ferric uptake regulator protein (Fur), a protein classically defined as a transcriptional repressor. Recently, however, microarray studies have identified a number of genes in N. meningitidis that are iron and Fur activated, demonstrating a new role for Fur as a transcriptional activator. Since Fur has been shown to indirectly activate gene transcription through the repression of small regulatory RNA molecules in other organisms, we hypothesized that a similar mechanism could account for Fur-dependent, iron-activated gene transcription in N. meningitidis. In this study, we used a bioinformatics approach to screen for the presence of Fur-regulated small RNA molecules in N. meningitidis MC58. This screen identified one small RNA, herein named NrrF (for neisserial regulatory RNA responsive to iron [Fe]), which was demonstrated to be both iron responsive and Fur regulated and which has a well-conserved orthologue in N. gonorrhoeae. In addition, this screen identified a number of other likely, novel small RNA transcripts. Lastly, we utilized a new bioinformatics approach to predict regulatory targets of the NrrF small RNA. This analysis led to the identification of the sdhA and sdhC genes, which were subsequently demonstrated to be under NrrF regulation in an nrrF mutant. This study is the first report of small RNAs in N. meningitidis and the first to use a bioinformatics approach to identify, a priori, regulatory targets of a small RNA. PMID:17351036

  16. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation.

    PubMed

    Heanue, T A; Reshef, R; Davis, R J; Mardon, G; Oliver, G; Tomarev, S; Lassar, A B; Tabin, C J

    1999-12-15

    We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2). Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6. PMID:10617572

  17. Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation

    PubMed Central

    Heanue, Tiffany A.; Reshef, Ram; Davis, Richard J.; Mardon, Graeme; Oliver, Guillermo; Tomarev, Stanislav; Lassar, Andrew B.; Tabin, Clifford J.

    1999-01-01

    We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2). Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6. PMID:10617572

  18. Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vibrio parahaemolyticus, a marine bacterium, is the causative agent of gastroenteritis associated with the consumption of seafood. It contains a homologue of the toxRS operon that in V. cholerae is the key regulator of virulence gene expression. We examined a non-polar mutation in toxRS to determi...

  19. Appetitive Cue-Evoked ERK Signaling in the Nucleus Accumbens Requires NMDA and D1 Dopamine Receptor Activation and Regulates CREB Phosphorylation

    ERIC Educational Resources Information Center

    Kirschmann, Erin K. Z.; Mauna, Jocelyn C.; Willis, Cory M.; Foster, Rebecca L.; Chipman, Amanda M.; Thiels, Edda

    2014-01-01

    Conditioned stimuli (CS) can modulate reward-seeking behavior. This modulatory effect can be maladaptive and has been implicated in excessive reward seeking and relapse to drug addiction. We previously demonstrated that exposure to an appetitive CS causes an increase in the activation of extracellular signal-regulated kinase (ERK) and cyclic-AMP…

  20. The requirement of c-Jun N-terminal kinase 2 in regulation of hypoxia-inducing factor-1α mRNA stability.

    PubMed

    Zhang, Dongyun; Li, Jingxia; Zhang, Min; Gao, Guangxun; Zuo, Zhenghong; Yu, Yonghui; Zhu, Linda; Gao, Jimin; Huang, Chuanshu

    2012-10-01

    The mRNA of hif-1α is considered as being constitutively and ubiquitously expressed, regardless of the level of oxygen tension. However many recent reports have showed that hif-1α mRNA could be regulated by natural antisense transcripts, potential microRNAs, and low O(2). In this study, it was found that a deficiency of JNK2 expression reduced HIF-1α protein induction in response to nickel treatment resulting from the impaired expression of hif-1α mRNA. Both the promoter luciferase assay and mRNA degradation assay clearly showed that depletion of JNK2 affected stability of hif-1α mRNA, rather than regulated its transcription. In addition, nucleolin, a classic histone chaperone, was demonstrated to physically bind to hif-1α mRNA and maintain its stability. Further investigation indicated that JNK2 regulated nucleolin expression and might in turn stabilize hif-1α mRNA. Collectively, we provided one more piece of evidence for the oncogenic role of JNK2 and nucleolin in regulating the cancer microenvironments by controlling HIF-1α expression. PMID:22910906